Beta-carbolines useful for treating inflammatory disease

ABSTRACT

This invention provides beta-carboline compounds of formula 
     
       
         
         
             
             
         
       
         
         
           
             wherein Ring A is a substituted pyridinyl, pyrimidinyl, morpholinyl, piperidinyl, piperazinyl, pyrrolidinyl, pyranyl, tetrahydrofuranyl, cyclohexyl, cyclopentyl or thiomorpholinyl ring and R 1 , R 2  and R 3  are as described in the specification. The compounds are IKK-2 inhibitors that are useful for treating IKK-2-mediated diseases such as inflammatory diseases and cancer.

RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.10/821,545, filed Apr. 9, 2004 (pending), which claims the benefit ofU.S. Provisional Application Ser. No. 60/461,468, filed Apr. 9, 2003(abandoned). The entire contents of each of the above-referenced patentapplications are incorporated herein by this reference.

FIELD OF THE INVENTION

This invention relates to beta-carboline compounds, pharmaceuticalcompositions thereof, and methods of using the compositions for treatingdisease. The compounds are particularly useful for treating inflammatorydisease and cancer.

BACKGROUND OF THE INVENTION

The transcription (nuclear) factor NF-κB is a member of the Rel proteinfamily, and is typically a heterodimer composed of p50 and p65 subunits.NF-κB is constitutively present in the cytosol, and is inactivated byits association with one of the IκB family of inhibitors. Palombella etal., WO 95/25533, teaches that the ubiquitin-proteasome pathway plays anessential role in the regulation of NF-κB activity, being responsiblefor the processing of p105 to p50 and the degradation of the inhibitorprotein IκB-α. Chen et al., Cell 84:853 (1996), teaches that prior todegradation, IκB-α undergoes selective phosphorylation at serineresidues 32 and 36 by the multisubunit IκB kinase complex (IKK). IκB-αis phosphorylated by IKK, which has two catalytic subunits, IKK-1 (IκBkinase a or IKK-α) and IKK-2 (IκB kinase β or IKK-β). Oncephosphorylated, IκB is targeted for ubiquitination and degradation bythe 26S proteasome, allowing translocation of NF-κB into the nucleus,where it binds to specific DNA sequences in the promoters of targetgenes and stimulates their transcription. Inhibitors of IKK can blockthe phosphorylation of IκB and its further downstream effects,particularly those associated with NF-κB transcription factors.

The protein products of genes under the regulatory control of NF-κBinclude cytokines, chemokines, cell adhesion molecules, and proteinsmediating cellular growth and control. Importantly, many of theseproinflammatory proteins also are able to act, either in an autocrine orparacrine fashion, to further stimulate NF-κB activation. In addition,NF-κB plays a role in the growth of normal and malignant cells.Furthermore, NF-κB is a heterodimeric transcription factor which canactivate a large number of genes which code, inter alia, forproinflammatory cytokines such as IL-1, IL-2, TNFα or IL-6. NF-κB ispresent in the cytosol of cells, building a complex with its naturallyoccurring inhibitor IκB. The stimulation of cells, for example bycytokines, leads to the phosphorylation and subsequent proteolyticdegradation of IκB. This proteolytic degradation leads to the activationof NF-κB, which subsequently migrates into the nucleus of the cell andactivates a large number of proinflammatory genes.

Rinehart et al., U.S. Pat. No. 4,631,149 (1986), disclosesbeta-carboline compounds useful as antiviral, antibacterial, andantitumor agents.

Ritzeler et al., WO 01/68648, discloses beta-carboline compounds withIκB kinase inhibitory activity for use in the treatment of inflammatorydisorders (e.g., rheumatoid arthritis), osteoarthritis, asthma, cardiacinfarct, Alzheimer's disease, carcinomatous disorders (potentiation ofcytotoxic therapies) and atherosclerosis.

It would be beneficial to provide novel IKK inhibitors that possess goodtherapeutic properties, especially for the treatment of inflammatorydisease.

DESCRIPTION OF THE INVENTION

This invention provides compounds that are useful for treatingIKK-2-mediated diseases, such as inflammatory diseases and cancer. Thecompounds are represented by formula I:

wherein Ring A is selected from the group consisting of:

(a) a pyridinyl or pyrimidinyl ring that is substituted by (i)—CH₂C(O)-G and 0-1 R^(6a) or (ii) 1-2 R^(6a), and

(b) a morpholinyl, piperidinyl, piperazinyl, pyrrolidinyl, pyranyl,tetrahydrofuranyl, cyclohexyl, cyclopentyl or thiomorpholinyl ring thatis substituted by (i) —C(R⁹)₃, —W-G, or -G, (ii) 0-4 R^(6b) and (iii)0-1 oxo groups on a ring carbon or 0-2 oxo groups on a ring sulfur;

each R^(6a) is independently selected from C₁₋₆ aliphatic, halo, C₁₋₆alkoxy, or amino;

each R^(6b) is independently selected from C₁₋₃ aliphatic or —N(R⁷)₂,and two R^(6b) on the same or an adjacent carbon optionally are takentogether with the intervening carbon(s) to form a 5-6 membered ringhaving 1-2 ring heteroatoms selected from N, O or S;

W is -Q-, -Q-C(O)—, —C(R⁹)₂—C(R⁹)(R¹²)—, or —C(R⁹)₂-[C(R⁹)(R¹²)]₂—;

Q is —C(R⁹)₂— or —C(R⁹)₂C(R⁹)₂—;

G is —OH, —NR⁴R⁵, —N(R⁹)CONR⁴R⁵, —N(R⁹)SO₂(C₁₋₃ aliphatic), —N(R⁹)COCF₃,—N(R⁹)CO(C₁₋₆ aliphatic), —N(R⁹)CO(heterocyclyl), —N(R⁹)CO(heteroaryl),—N(R⁹)CO(aryl), a 3-7 membered heterocyclyl ring, or a 5-6 memberedheteroaryl, wherein each of the heteroaryl, aryl and heterocyclylmoieties of G is optionally substituted by 1-3 R¹⁰;

R¹ is hydrogen, halo, C₁₋₃ aliphatic, amino, cyano, (C₁₋₃ alkyl)₁₋₂amino, C₁₋₃ alkoxy, —CONH₂, —NHCOCF₃, or —CH₂NH₂;

R² is hydrogen, halo, C₁₋₃ aliphatic, —CF₃;

R³ is hydrogen, halo, C₁₋₆ aliphatic, C₁₋₆ haloalkyl, C₁₋₆ alkoxy,hydroxy, amino, cyano, or (C₁₋₆ alkyl)₁₋₂ amino;

R⁴ is hydrogen, 3-7 membered heterocyclyl, or C₁₋₆ aliphatic;

R⁵ is hydrogen, C₁₋₆ aliphatic group or a 3-7 membered heterocyclic ringhaving 1-2 ring heteroatoms selected from N, O, or S, wherein R⁵ isoptionally substituted by halo, —OR⁷, —CN, —SR⁸, —S(O)₂R⁸, —S(O)₂N(R⁷)₂,—C(O)R⁷, —CO₂R⁷, —N(R⁷)₂, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)CO₂R⁸, or—N(R⁷)C(O)N(R⁷)₂;

each R⁷ is independently selected from hydrogen or C₁₋₄ aliphatic, ortwo R⁷ on the same nitrogen atom are taken together with the nitrogen toform a 5-6 membered heteroaryl or heterocyclyl ring;

each R⁸ is independently selected from C₁₋₄ aliphatic;

each R⁹ is independently selected from hydrogen or C₁₋₃ aliphatic;

each R¹⁰ is independently selected from oxo, —R¹¹, -T-R¹¹, or -V-T-R¹¹;

each R¹¹ is independently selected from C₁₋₆ aliphatic, halo,—S(O)₂N(R⁷)₂, —OR⁷, —CN, —SR⁸, —S(O)₂R⁸, —C(O)R⁷, —CO₂R⁷, —N(R⁷)₂,—C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)CO₂R⁷, or —N(R⁷)C(O)N(R⁷)₂;

T is a straight or branched C₁₋₄ alkylene chain;

V is —O—, —N(R⁷)—, —S—, —S(O)—, —S(O)₂—, —C(O)—, or —CO₂—; and

R¹² is hydrogen or an amino acid side chain.

The term “aliphatic” as used herein means straight-chain, branched orcyclic C₁-C₁₂ hydrocarbons which are completely saturated or whichcontain one or more units of unsaturation but which are not aromatic.For example, suitable aliphatic groups include substituted orunsubstituted linear, branched or cyclic alkyl, alkenyl, alkynyl groupsand hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or(cycloalkyl)alkenyl. The terms “alkyl”, “alkoxy”, “hydroxyalkyl”,“alkoxyalkyl”, and “alkoxycarbonyl”, used alone or as part of a largermoiety, include both straight and branched chains containing one totwelve carbon atoms. The terms “alkenyl” and “alkynyl”, used alone or aspart of a larger moiety, include both straight and branched chainscontaining two to twelve carbon atoms. The term “cycloalkyl, used aloneor as part of a larger moiety, includes cyclic C₃-C₁₂ hydrocarbons whichare completely saturated or which contain one or more units ofunsaturation, but which are not aromatic.

The terms “haloalkyl”, “haloalkenyl” and “haloalkoxy”, mean alkyl,alkenyl or alkoxy, as the case may be, substituted with one or morehalogen atoms. The term “halogen” means F, Cl, Br, or I.

The term “heteroatom” means nitrogen, oxygen, or sulfur and includes anyoxidized form of nitrogen and sulfur, and the quaternized form of anybasic nitrogen. Also the term “nitrogen” includes a substitutablenitrogen of a heterocyclic ring. As an example, in a saturated orpartially unsaturated ring having 0-3 heteroatoms selected from oxygen,sulfur or nitrogen, the nitrogen may be N (as in3,4-dihydro-2-pyrrolyl), NH (as in pyrrolidinyl) or NR⁺ (as inN-substituted pyrrolidinyl).

The terms “carbocycle”, “carbocyclyl”, “carbocyclo”, or “carbocyclic” asused herein means an aliphatic ring system having three to fourteenmembers.

The terms “carbocycle”, “carbocyclyl”, “carbocyclo”, or “carbocyclic”whether saturated or partially unsaturated, also refers to rings thatare optionally substituted. The terms “carbocycle”, “carbocyclyl”,“carbocyclo”, or “carbocyclic” also include aliphatic rings that arefused to one or more aromatic or nonaromatic rings, such as in adecahydronaphthyl or tetrahydronaphthyl, where the radical or point ofattachment is on the aliphatic ring.

The term “aryl” used alone or as part of a larger moiety as in“aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to aromatic ring groupshaving five to fourteen members, such as phenyl, benzyl, phenethyl,1-naphthyl, 2-naphthyl, 1-anthracyl and 2-anthracyl. The term “aryl”also refers to rings that are optionally substituted. The term “aryl”may be used interchangeably with the term “aryl ring”. “Aryl” alsoincludes fused polycyclic aromatic ring systems in which an aromaticring is fused to one or more rings. Examples include 1-naphthyl,2-naphthyl, 1-anthracyl and 2-anthracyl. Also included within the scopeof the term “aryl”, as it is used herein, is a group in which anaromatic ring is fused to one or more non-aromatic rings, such as in anindanyl, phenanthridinyl, or tetrahydronaphthyl, where the radical orpoint of attachment is on the aromatic ring.

The term “heterocycle”, “heterocyclyl”, or “heterocyclic” as used hereinincludes non-aromatic ring systems having five to fourteen members,preferably five to ten, in which one or more ring carbons, preferablyone to four, are each replaced by a heteroatom such as N, O, or S.Examples of heterocyclic rings include 3-1H-benzimidazol-2-one,(1-substituted)-2-oxo-benzimidazol-3-yl, 2-tetrahydrofuranyl,3-tetrahydrofuranyl, 2-tetrahydropyranyl, 3-tetrahydropyranyl,4-tetrahydropyranyl, [1,3]-dioxalanyl, [1,3]-dithiolanyl,[1,3]-dioxanyl, 2-tetrahydrothiophenyl, 3-tetrahydrothiophenyl,2-morpholinyl, 3-morpholinyl, 4-morpholinyl, 2-thiomorpholinyl,3-thiomorpholinyl, 4-thiomorpholinyl, 1-pyrrolidinyl, 2-pyrrolidinyl,3-pyrrolidinyl, 1-piperazinyl, 2-piperazinyl, 1-piperidinyl,2-piperidinyl, 3-piperidinyl, 4-piperidinyl, 4-thiazolidinyl,diazolonyl, N-substituted diazolonyl, 1-phthalimidinyl, benzoxanyl,benzopyrrolidinyl, benzopiperidinyl, benzoxolanyl, benzothiolanyl, andbenzothianyl. Also included within the scope of the term “heterocyclyl”or “heterocyclic”, as it is used herein, is a group in which anon-aromatic heteroatom-containing ring is fused to one or more aromaticor non-aromatic rings, such as in an indolinyl, chromanyl,phenanthridinyl, or tetrahydroquinolinyl, where the radical or point ofattachment is on the non-aromatic heteroatom-containing ring. The term“heterocycle”, “heterocyclyl”, or “heterocyclic” whether saturated orpartially unsaturated, also refers to rings that are optionallysubstituted.

The term “heteroaryl”, used alone or as part of a larger moiety as in“heteroaralkyl” or “heteroarylalkoxy”, refers to heteroaromatic ringgroups having five to fourteen members. Examples of heteroaryl ringsinclude 2-furanyl, 3-furanyl, 3-furazanyl, N-imidazolyl, 2-imidazolyl,4-imidazolyl, 5-imidazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl,2-oxadiazolyl, 5-oxadiazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl,1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 1-pyrazolyl, 2-pyrazolyl,3-pyrazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl,5-pyrimidyl, 3-pyridazinyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl,5-tetrazolyl, 2-triazolyl, 5-triazolyl, 2-thienyl, 3-thienyl,carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl,quinolinyl, benzotriazolyl, benzothiazolyl, benzooxazolyl,benzimidazolyl, isoquinolinyl, indazolyl, isoindolyl, acridinyl, orbenzoisoxazolyl. Also included within the scope of the term“heteroaryl”, as it is used herein, is a group in which a heteroatomicring is fused to one or more aromatic or nonaromatic rings where theradical or point of attachment is on the heteroaromatic ring. Examplesinclude tetrahydroquinolinyl, tetrahydroisoquinolinyl, andpyrido[3,4-d]pyrimidinyl. The term “heteroaryl” also refers to ringsthat are optionally substituted. The term “heteroaryl” may be usedinterchangeably with the term “heteroaryl ring” or the term“heteroaromatic”.

An aryl (including aralkyl, aralkoxy, aryloxyalkyl and the like) orheteroaryl (including heteroaralkyl and heteroarylalkoxy and the like)group may contain one or more substituents. Examples of suitablesubstituents on the unsaturated carbon atom of an aryl, heteroaryl,aralkyl, or heteroaralkyl group include a halogen, —R^(O), —OR^(O),—SR^(O), 1,2-methylene-dioxy, 1,2-ethylenedioxy, protected OH (such asacyloxy), phenyl (Ph), substituted Ph, —O(Ph), substituted —O(Ph),—CH₂(Ph), substituted —CH₂(Ph), —CH₂CH₂(Ph), substituted —CH₂CH₂(Ph),—NO₂, —CN, —N(R^(O))₂, —NR^(O)C(O)R^(O), —NR^(O)C(O)N(R^(O))₂,—NR^(O)CO₂R^(O), —NR^(O)NR^(O)C(O)R^(O), —NR^(O)NR^(O)C(O)N(R^(O))₂,—NR^(O)NR^(O)CO₂R^(O), —C(O)C(O)R^(O), —C(O)CH₂C(O)R^(O), —CO₂R^(O),—C(O)R^(O), —-C(O)N(R^(O))₂, —OC(O)N(R^(O))₂, —S(O)₂R^(O),—SO₂N)(R^(O))₂, —S(O)R^(O), —NR^(O)SO₂N)(R^(O))₂, —NR^(O)SO₂R^(O),—C(═S)N(R^(O)—C(═NH)—N(R^(O))₂, —(CH₂)_(y)NHC(O)R^(O), —(CH₂)_(y)NHC(O)CH(V—R^(O))(R^(O)); wherein each R^(O) is independently selected fromhydrogen, a substituted or unsubstituted aliphatic group, anunsubstituted heteroaryl or heterocyclic ring, phenyl (Ph), substitutedPh, —O(Ph), substituted —O(Ph), —CH₂(Ph), or substituted —CH₂(Ph); y is0-6; and V is a linker group. Examples of substituents on the aliphaticgroup or the phenyl ring of R^(O) include amino, alkylamino,dialkylamino, aminocarbonyl, halogen, alkyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylaminocarbonyloxy, dialkylaminocarbonyloxy,alkoxy, nitro, cyano, carboxy, alkoxycarbonyl, alkylcarbonyl, hydroxy,haloalkoxy, or haloalkyl.

An aliphatic group or a non-aromatic heterocyclic ring may contain oneor more substituents. Examples of suitable substituents on the saturatedcarbon 20 of an aliphatic group or of a non-aromatic heterocyclic ringinclude those listed above for the unsaturated carbon of an aryl orheteroaryl group and the following: ═O, ═S, ═NNHR*, ═NN(R*)₂, ═N—,═NNHC(O)R*, ═NNHCO₂(alkyl), ═NNHSO₂(alkyl), or ═NR*, where each R* isindependently selected from hydrogen, an unsubstituted aliphatic groupor a substituted aliphatic group. Examples of substituents on thealiphatic group include amino, alkylamino, dialkylamino, aminocarbonyl,halogen, alkyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylaminocarbonyloxy, dialkylaminocarbonyloxy, alkoxy, nitro, cyano,carboxy, alkoxycarbonyl, alkylcarbonyl, hydroxy, haloalkoxy, orhaloalkyl.

Suitable substituents on the nitrogen of a non-aromatic heterocyclicring include —R⁺, —N(R⁺)₂, —C(O)R⁺, —CO₂R⁺, —C(O)C(O)R⁺, —C(O)CH₂C(O)R⁺,—SO₂R⁺, —SO₂N(R⁺)₂, —C(═S)N(R⁺)₂, —C(═NH)—N(R⁺)₂, and —NR⁺SO₂R⁺; whereineach R⁺ is independently selected from hydrogen, an aliphatic group, asubstituted aliphatic group, phenyl (Ph), substituted Ph, —O(Ph),substituted —O(Ph), CH₂(Ph), substituted CH₂(Ph), or an unsubstitutedheteroaryl or heterocyclic ring. Examples of substituents on thealiphatic group or the phenyl ring include amino, alkylamino,dialkylamino, aminocarbonyl, halogen, alkyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylaminocarbonyloxy, dialkylaminocarbonyloxy,alkoxy, nitro, cyano, carboxy, alkoxycarbonyl, alkylcarbonyl, hydroxy,haloalkoxy, or haloalkyl.

The term “linker group” or “linker” means an organic moiety thatconnects two parts of a compound. Linkers are typically comprised of anatom such as oxygen or sulfur, a unit such as —NH—, —CH₂—, —C(O)—,—C(O)NH—, or a chain of atoms, such as an alkylidene chain. Themolecular mass of a linker is typically in the range of about 14 to 200,preferably in the range of 14 to 96 with a length of up to about sixatoms. Examples of linkers include a saturated or unsaturated C₁₋₆alkylidene chain which is optionally substituted, and wherein one or twosaturated carbons of the chain are optionally replaced by —C(O)—,—C(O)C(O)—, —CONH—, —CONHNH—, —CO₂—, —OC(O)—, —NHCO₂—, —O—, —NHCONH—,—OC(O)NH—, —NHNH—, —NHCO—, —S—, —SO—, —SO₂—, —NH—, —SO₂NH—, or —NHSO₂—.

The term “alkylidene chain” or “alkylene chain” refers to an optionallysubstituted, straight or branched carbon chain that may be fullysaturated or have one or more units of unsaturation. The optionalsubstituents are as described above for an aliphatic group.

A combination of substituents or variables is permissible only if such acombination results in a stable or chemically feasible compound. Astable compound or chemically feasible compound is one in which thechemical structure is not substantially altered when kept at atemperature of 40° C. or less, in the absence of moisture or otherchemically reactive conditions, for at least a week.

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure; i.e., the R and Sconfigurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds are within the scope of the invention.Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen atom by a deuteriumor tritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enrichedcarbon are within the scope of this invention.

In one embodiment of the invention, Ring A is selected from a pyridinylor a pyrimidinyl ring that is substituted by 1-2 R^(6a) groups. In thisembodiment, preferred Ring A include a 3-pyridinyl or a 5-pyrimidinylring, shown below by the compounds of formula II-A and II-B,respectively.

Preferably, R^(6a) is selected from halo or a C₁₋₆ aliphatic, such aschloro or methyl. When R^(6a) is an aliphatic group such as methyl, afavorable position for the R^(6a) group is at the 2-position of thepyridinyl ring or the 4-position of the pyrimidinyl ring, as shown inII-C above. Particular Ring A moieties are 2-methyl-3-pyridinyl and4-methyl-5-pyrimidinyl. It has been found that compounds of formula II-Cwhere R^(6a) is a methyl group are surprisingly more potent inbiological testing for IKK inhibition than analogous compounds that havean unsubstituted Ring A pyridine, such as those described in theaforementioned Ritzeler et al. PCT application WO 01/68648.

Preferred R¹ groups are small groups such as hydrogen, methyl, amino andfluoro.

Preferred R² groups include hydrogen and halo. Chloro is a preferred R²halo group.

Preferred R³ groups include hydrogen, halo (especially chloro) andalkoxy. Examples of suitable alkoxy groups include C_(1-d) alkoxy groupssuch as methoxy, ethoxy, propoxy and cyclopropylmethoxy.

In another embodiment, Ring A is selected from a 5-6 memberednon-aromatic ring having 0-2 ring heteroatoms selected from nitrogen,oxygen and sulfur. These are designated generally as compounds offormula III. Examples of non-aromatic Ring A groups include amorpholinyl, piperidinyl, piperazinyl, pyrrolidinyl, pyranyl,tetrahydrofuranyl, cyclohexyl, cyclopentyl and a thiomorpholinyl ring.Preferably, such non-aromatic rings are substituted by (i) —C(R⁹)₃ or—W-G, (ii) 0-4 R^(6b) and (iii) 0-1 oxo groups on a ring carbon or 0-2oxo groups on a ring sulfur. More preferably, such non-aromatic ringsare substituted by (i) —W-G, (ii) 0-2 R^(6b) and (iii) 0-1 oxo groups ona ring carbon or 0-2 oxo groups on a ring sulfur.

A preferred G is —NR⁴R⁵ or a 3-7 membered heterocyclyl ring. Morepreferably G is —NR⁴R⁵ or a 5-6 membered heterocyclyl ring, where G issubstituted by 1-2 R¹⁰.

A preferred R⁴ is a hydrogen, 5-6 membered heterocyclyl ring, or C₁₋₆aliphatic, more preferably hydrogen or C₁₋₆ aliphatic. R⁴ may also be aC₁₋₆alkoxy.

A preferred R⁵ is a hydrogen, 5-6 membered heterocyclyl ring, or C₁₋₆aliphatic, more preferably hydrogen or C₁₋₆ aliphatic.

Various formula III compounds where Ring A is a non-aromatic ring areshown in Table 2. For ease of viewing, substituents on thesenon-aromatic Ring A compounds, except for the oxo group in some cases,are not shown.

TABLE 2 Compounds where Ring A is Non-Aromatic III-A

III-B

III-C

III-D

III-E

III-F

III-G

III-H

III-J

III-K

III-L

III-M

When Ring A is a non-aromatic, six-membered heterocylic ring, afavorable position for the —W-G and —C(R⁹)₃ substituents on Ring A isortho to the position where the beta-carboline portion is attached. Forexample, in compounds III-A, III-B, III-D, III-H, III-J and III-M, apreferred position for attachment of —W-G and —C(R⁹)₃ is at the Ring Anitrogen or at N−1 in the case of compound

Preferably, W is -Q-, -Q-C(O)—, —C(R⁹)₂—C(R⁹)(R¹²)—, or—C(R⁹)₂—[C(R⁹)(R¹²)]₂— where R⁹ is hydrogen. More preferably, W is -Q-,-Q-C(O)—, or —C(R⁹)₂—C(R⁹)(R¹²)—. R¹² is hydrogen, C₁₋₆ aliphatic,substituted or unsubstituted phenyl, substituted or unsubstituted benzylor an amino acid side chain, particularly the side chain of a naturalamino acid. Examples of particular natural amino acids include alanine,phenylalanine, valine, leucine, isoleucine, serine, tyrosine, asparticacid and glutamic acid.

In one embodiment, W is Q-C(O)—. In this embodiment, a preferred Q is—CH₂— or —CH₂—CH₂—, more preferably —CH₂—.

In another embodiment, Ring A is substituted by 0-2 R^(6b). A preferredR^(6b) group is methyl. When Ring A is a non-aromatic six membered ring,one embodiment provides compounds of formula III where there are twomethyl groups on the Ring A position para to the position where the betacarboline portion is attached. An example of this embodiment is acompound where Ring A is a 6,6-dimethyl-morpholinyl ring. Preferably,such compounds are further substituted by —W-G as described above.

When Ring A is a morpholinyl ring, it has been found that compoundshaving the “S” stereochemistry at position 3 of the morpholine ring arepreferred, as shown below by compounds of formula (S)-III-A.

where n is 0-4 and R¹, R², R³, W, G and R^(6b) are as defined above. Byanalogy, it is expected that “S” stereochemistry is also preferred forother six-membered non-aromatic Ring A compounds of formula III.

One embodiment relates to compounds of formula III-A or (S)-III-A whereR¹ is hydrogen, halo, methyl or amino;

R² is hydrogen, methyl or halo;

R³ is hydrogen, halo, alkoxy, or (C₁₋₆ aliphatic)₂ amino;

Ring A is substituted by 0-2 R^(6b);

R^(6b) is C₁₋₃ aliphatic;

W is -Q-, -Q-C(O)—, —C(R⁹)₂—C(R⁹)(R¹²) or —C(R⁹)₂—[C(R⁹)(R¹²)]₂—;

Q is —C(R⁹)₂— or —C(R⁹)₂C(R⁹)₂—;

G is —NR⁴R⁵, —N(R⁹)C(O)NR⁴R⁵, —N(R⁹)SO₂(C₁₋₃ aliphatic), —N(R⁹)C(O)CF₃,—N(R⁹)CO(C₁₋₆ aliphatic), and —N(R⁹)CO(heterocyclyl),—N(R⁹)CO(heteroaryl), —N(R⁹)CO(aryl), a 5-6 membered heterocyclyl ring,or a 5-6 membered heteroaryl, wherein each of the heteroaryl, aryl andheterocyclyl moieties of G is optionally substituted by 1-3 R¹⁰;

R⁴ is hydrogen or C₁₋₆ aliphatic;

R⁵ is hydrogen or a C₁₋₆ aliphatic group that is optionally substitutedby halo, —OR⁷, —CN, —SR⁸, —S(O)₂R⁸, —S(O)₂N(R⁷)₂, —C(O)R⁷, —CO₂R⁷,—N(R⁷)₂, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)CO₂R⁸, or —N(R⁷)C(O)N(R⁷)₂;

each R⁷ is independently selected from hydrogen or C₁₋₄ aliphatic, ortwo R⁷ on the same nitrogen atom are taken, together with the nitrogento form a 5-6 membered heteroaryl or heterocyclyl ring;

each R⁸ is independently selected from C₁₋₄ aliphatic;

R⁹ is hydrogen;

each R¹⁰ is independently selected from oxo, R¹¹, T-R¹¹, or V-T-R¹¹;

each R¹¹ is independently selected from C₁₋₆ aliphatic, halo,—S(O)₂N(R⁷)₂, —OR⁷, —CN, —SR⁸, —S(O)₂R⁸, —C(O)R⁷, —CO₂R⁷, —N(R⁷)₂,—C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)CO₂R⁷, or —N(R⁷)C(O)N(R⁷)₂;

T is a straight or branched C₁₋₄ alkylene chain;

V is —O—, —N(R⁷)—, —S—, —S(O)—, —S(O)₂—, —C(O)—, or —CO₂—; and

R¹² is hydrogen, C₁₋₆ aliphatic, substituted or unsubstituted phenyl, orsubstituted or unsubstituted benzyl.

Another embodiment relates to compounds of formula III-A or (S)-III-Awhere:

R¹ is hydrogen, methyl, fluoro or amino;

R² is chloro;

R³ is hydrogen or alkoxy;

Ring A is substituted by —W-G and 0-2 R^(6b);

R^(6b) is methyl;

W is -Q-, -Q-C(O)— or —C(R⁹)₂—C(R⁹)(R¹²);

Q is —C(R⁹)₂— or —C(R⁹)₂C(R⁹)₂—;

G is —NR⁴R⁵, —N(R⁹)C(O)NR⁴R⁵, —N(R⁹)C(O)CF₃, —N(R⁹)CO(C₁₋₆ aliphatic),and —N(R⁹)CO(heterocyclyl) —N(R⁹)CO(heteroaryl), a 5-6 memberedheterocyclyl ring, or a 5-6 membered heteroaryl, wherein each of theheteroaryl and heterocyclyl moieties of G is optionally substituted by1-3 R¹⁰;

R⁴ is hydrogen or C₁₋₆ aliphatic;

R⁵ is hydrogen or C₁₋₆ aliphatic;

each R⁷ is independently selected from hydrogen or C₁₋₄ aliphatic, ortwo R⁷ on the same nitrogen atom are taken together with the nitrogen toform a 5-6 membered heteroaryl or heterocyclyl ring;

each R⁹ is independently selected from C₁₋₄ aliphatic;

R⁹ is hydrogen;

each R¹⁰ is independently selected from oxo, R¹¹, T-R¹¹, or V-T-R¹¹;

each R¹¹ is independently selected from C₁₋₆ aliphatic, halo,—S(O)₂N(R⁷)₂, —OR⁷—CN, —SR⁸, —S(O)₂R⁸, —C(O)R⁷, —CO₂R⁷, —N(R⁷)₂,—C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)CO₂R⁷, or —N(R⁷)C(O)N(R⁷)₂;

T is a straight or branched C₁₋₄ alkylene chain;

V is —O—, —N(R⁷)—, —S—, —S(O)—, —S(O)₂—, —C(O)—, or —CO₂—; and

R¹² is hydrogen, C₁₋₆ aliphatic, phenyl, or benzyl.

Preferred compounds of formula III-A are the compounds of formula(S)-III-A′:

where R¹, R², R³, W and G are as defined above for (S)-III-A.

Another embodiment relates to compounds of formula III-A-a:

or a pharmaceutically acceptable salt thereof, wherein:

Q is —CH₂—, —CH(R⁹)—, or —C(R⁹)₂—;

G is —NR⁴R⁵ or a 3-7 membered heterocyclyl or heteroaryl ring that isoptionally substituted by 1-2 R¹⁰;

R¹ is hydrogen, halo, C₁₋₃ aliphatic, amino, cyano, (C₁₋₃ alkyl)₁₋₂amino, C₁₋₃ alkoxy, (C₁₋₃ aliphatic)-C(O)—, (C₁₋₆ aliphatic)-CO₂—, or(C₁₋₃ aliphatic)-C(O)NH—;

R² is hydrogen, halo, C₁₋₃ aliphatic, C₁₋₃ alkoxy, C₁₋₃ haloalkoxy, orC₁₋₃ haloalkyl;

R³ is hydrogen, halo, C₁₋₆ aliphatic, C₁₋₆ haloalkyl, C₁₋₆ alkoxy,hydroxy, amino, cyano, or (C₁₋₆ alkyl)₁₋₂-amino;

R⁴ is hydrogen or C₁₋₆ aliphatic;

R⁵ is a C₁₋₆ aliphatic group that is optionally substituted by halo,—OR⁷, —CN, —SR⁸, —S(O)₂R⁸, —C(O)R⁷, —CO₂R⁷, —N(R⁷)₂, —C(O)N(R⁷)₂,—N(R⁷)C(O)R⁷, —N(R⁷)CO₂R⁸, or —N(R⁷)C(O)N(R⁷)₂;

Ring A is substituted by 0-4 R^(6b);

each R^(6b) is independently selected from a C₁₋₆ aliphatic group;

each R⁷ is independently selected from hydrogen or C₁₋₄ aliphatic, ortwo R⁷ on the same nitrogen atom are taken together with the nitrogen toform a 5-6 membered heteroaryl or heterocyclyl ring;

each R⁸ is independently selected from C₁₋₄ aliphatic;

each R⁹ is independently selected from a C₁₋₃ aliphatic;

each R¹⁰ is independently selected from R¹¹, T-R¹¹, or V-T-R¹¹;

each R¹¹ is independently selected from C₁₋₆ aliphatic, halo, —OR⁷, —CN,—SR⁸, —S(O)—C(O)R⁷, —CO₂R⁷, —N(R⁷)₂, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,—N(R⁷)CO₂R⁷, or —N(R⁷)C(O)N(R⁷)₂;

T is a straight or branched C₁₋₄ alkylene chain; and

V is —O—, —N(R⁷)—, —S—, —S(O)—, —S(O)₂—, —C(O)—, or —CO₂—.

One embodiment relates to compounds of formula III-A-a where:

Q is —CH₂—, or —CH(R⁹)—;

G is —NR⁴R⁵ or a 5-6 membered heterocyclyl or heteroaryl ring that isoptionally substituted by 1-2 R^(n);

R¹ is hydrogen, halo, C₁₋₂ alkyl, amino, or (C₁₋₂ alkyl)₁₋₂ amino;

R² is hydrogen, halo, C₁₋₂ aliphatic, C₁₋₂ alkoxy, or C₁₋₂ haloalkyl;

R³ is hydrogen, halo, C₁₋₂ aliphatic, C₁₋₂ alkoxy, or C₁₋₂ haloalkyl;

R⁴ is hydrogen or C₁₋₆ aliphatic;

R⁵ is a C₁₋₆ aliphatic group that is optionally substituted by halo,—OR⁷, —CN, —SR⁸, —S(O)₂R⁹, —C(O)R⁷, —CO₂R⁷, —N(R⁷)₂, —C(O)N(R⁷)₂,—N(R⁷)C(O)R⁷, —N(R⁷)CO₂R⁸, or —N(R⁷)C(O)N(R⁷)₂;

Ring A is substituted by 0-2 R^(6b);

each R^(6b) is independently selected from a C₁₋₃ aliphatic group;

each R⁷ is independently selected from hydrogen or C₁₋₄ aliphatic, ortwo R⁷ on the same nitrogen atom are taken together with the nitrogen toform a 5-6 membered heteroaryl or heterocyclyl ring;

R⁸ is C₁₋₄ aliphatic;

R⁹ is independently selected from a C₁₋₃ aliphatic;

each R¹⁰ is independently selected from R¹¹, T-R¹¹, or V-T-R¹¹;

each R¹¹ is independently selected from C₁₋₆ aliphatic, halo, —OR⁷, —CN,—SR⁸, —S(O)₂R⁸, —C(O)R⁷, —CO₂R⁷, —N(R⁷)₂, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,—N(R⁷)CO₂R⁷, or —N(R⁷)C(O)N(R⁷)₂;

T is a straight or branched C₁₋₄ alkylene chain; and

V is —O—, —N(R⁷)—, —S—, —S(O)—, —S(O)₂—, —C(O)—, or —CO₂—.

Another embodiment relates to compounds of formula III-A-a where:

Q is —CH₂—, or —CH(R⁹)—;

G is —NR⁴R⁵ or a 5-6 membered heterocyclyl ring, having 1-2 ringheteroatoms selected from oxygen or nitrogen, that is optionallysubstituted by 1-2 R¹⁰;

R¹ is hydrogen, halo, methyl, amino, or (C₁₋₂ alkyl)₁₋₂ amino;

R² is hydrogen, halo, C₁₋₂ aliphatic, or C₁₋₂ haloalkyl;

R³ is hydrogen, halo, or C₁₋₂ aliphatic;

R⁴ is hydrogen or C₁₋₆ aliphatic;

R⁵ is a C₁₋₆ aliphatic group that is optionally substituted by halo,—OR⁷, —CN, —SR⁸, —S(O)₂R⁸, —C(O)R⁷, —CO₂R⁷, —N(R⁷)₂, —C(O)N(R⁷)₂,—N(R⁷)C(O)R⁷, —N(R⁷)CO₂R⁸, or —N(R⁷)C(O)N(R⁷)₂;

Ring A is substituted by zero or two R^(6b);

each R^(6b) is independently selected from a C₁₋₃ aliphatic group;

each R⁷ is independently selected from hydrogen or C₁₋₄ aliphatic, ortwo R⁷ on the same nitrogen atom are taken together with the nitrogen toform a 5-6 membered heteroaryl or heterocyclyl ring;

R⁸ is C₁₋₄ aliphatic;

R⁹ is independently selected from a C₁₋₃ aliphatic;

each R¹⁰ is independently selected from R¹¹, T-R¹¹, or V-T-R¹¹;

each R¹¹ is independently selected from C₁₋₆ aliphatic, halo, —OR⁷, —CN,—SR⁸, —S(O)₂R⁸, —C(O)R⁷, —CO₂R⁷, —N(R⁷)₂, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,—N(R⁷)CO₂O, or —N(R⁷)C(O)N(R⁷)₂;

T is a straight or branched C₁₋₄ alkylene chain; and

V is —O—, —N(R⁷)—, —S—, —S(O)—, —S(O)₂—, —C(O)—, or —CO₂—.

In preferred compounds of formula III-A-a, Q is —CH₂—; G is selectedfrom an optionally substituted piperidinyl, piperazinyl, morpholinyl,pyrrolidinyl, or —NR⁴R⁵; R⁴ is hydrogen or C₁₋₆ aliphatic; and R⁵ isC₁₋₆ aliphatic, 5-6 membered heterocyclyl, or C₁₋₆ hydroxyalkyl. Morepreferred are compounds where G is unsubstituted or substituted by 1-2groups independently selected from the group consisting of: C₁₋₃ alkyl,HO-alkyl, alkoxycarbonyl, mono- or dialkylaminocarbonyl, and HO₂C-alkyl.For compounds of formula III-A-a in each of the above embodiments, the(S) stereochemistry at the morpholine 3-position is preferred.

Another embodiment relates to compounds of formula III-A-aa:

or a pharmaceutically acceptable salt thereof wherein,

Q is —CH₂—, or —CH(R⁹)—;

G is —NR⁴R⁵ or a 3-7 membered heterocyclyl ring that is optionallysubstituted by 1-2 R¹⁰;

R¹ is hydrogen, halo, C₁₋₂ alkyl, amino, or (C₁₋₂ alkyl)₁₋₂ amino;

R² is hydrogen, halo, C₁₋₂ aliphatic, C₁₋₂ alkoxy, or C₁₋₂ haloalkyl;

R³ is hydrogen, halo, C₁₋₂ aliphatic, C₁₋₂ alkoxy, or C₁₋₂ haloalkyl;

R⁴ is hydrogen or C₁₋₆ aliphatic;

R⁵ is a C₁₋₆ aliphatic group that is optionally substituted by halo,—OR⁷, —CN, —SR⁸, —S(O)₂R⁸, —C(O)R⁷, —CO₂R⁷, —N(R⁷)₂, —C(O)N(R⁷)₂,—N(R⁷)C(O)R⁷, —N(R⁷)CO₂R⁸, or —N(R⁷)C(O)N(R⁷)₂;

each R^(6b) is independently selected from hydrogen or a C₁₋₆ aliphatic;

each R⁷ is independently selected from hydrogen or C₁₋₄ aliphatic, ortwo R⁷ on the same nitrogen atom are taken together with the nitrogen toform a 5-6 membered heteroaryl or heterocyclyl ring;

R⁸ is C₁₋₄ aliphatic;

R⁹ is independently selected from a C₁₋₃ aliphatic;

each R¹⁰ is independently selected from R¹¹, T-R¹¹, or V-T-R¹¹;

each R¹¹ is independently selected from C₁₋₆ aliphatic, halo, —OR⁷, —CN,—SR⁸, —S(O)₂R⁸, —C(O)R⁷, —CO₂R⁷, —N(R⁷)₂, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,—N(R⁷)CO₂R⁷, or —N(R⁷)C(O)N(R⁷)₂;

T is a straight or branched C₁₋₄ alkylene chain; and

V is —O—, —N(R⁷)—, —S—, —S(O)—, —S(O)₂—, —C(O)—, or

Another embodiment relates to compounds of formula III-A-aa where:

Q is —CH₂—;

G is —NR⁴R⁵ or a 5-6 membered heterocyclyl ring that is optionallysubstituted by 1-2 R¹¹;

R¹ is hydrogen, halo or methyl;

R² is hydrogen, halo, C₁₋₂ aliphatic, C₁₋₂ alkoxy, or C₁₋₂ haloalkyl;

R³ is hydrogen;

R⁴ is hydrogen or C₁₋₆ aliphatic;

R⁵ is a C₁₋₆ aliphatic group that is optionally substituted by halo,—OR⁷, —CN, —SR⁸, —S(O)₂R⁸, —C(O)R⁷, —CO₂R⁷, —N(R⁷)₂, —C(O)N(R⁷)₂,—N(R⁷)C(O)R⁷, —N(R⁷)CO₂R⁸, or —N(R⁷)C(O)N(R⁷)₂;

each R^(6b) is independently selected from hydrogen or a C₁₋₆ aliphatic;

each R⁷ is independently selected from hydrogen or C₁₋₄ aliphatic, ortwo R⁷ on the same nitrogen atom are taken together with the nitrogen toform a 5-6 membered heteroaryl or heterocyclyl ring;

R⁸ is C₁₋₄ aliphatic;

each R¹⁰ is independently selected from R¹¹, T-R¹¹, or V-T-R¹¹;

each R¹¹ is independently selected from C₁₋₆ aliphatic, halo, —OR⁷, —CN,—SR⁸, —S(O)₂R⁸, —C(O)R⁷, —CO₂R⁷, —N(R⁷)₂, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,—N(R⁷)CO₂R⁷, or —N(R⁷)C(O)N(R⁷)₂;

T is a straight or branched C₁₋₄ alkylene chain; and

V is —O—, —N(R⁷)—, —S—, —S(O)—, —S(O)₂—, —C(O)—, or —CO₂—.

Preferred compounds of III-A-aa are compounds where:

Q is —CH₂—;

G is selected from an optionally substituted piperidinyl, piperazinyl,morpholinyl, pyrrolidinyl, or —NR⁴R⁵;

R¹ is hydrogen, halo or methyl;

R² is halo;

R³ is hydrogen;

R⁴ is hydrogen or C₁₋₆ aliphatic;

R⁵ is C₁₋₆ alkoxy, C₁₋₆ aliphatic, or C₁₋₆ hydroxyalkyl;

each R^(6b) is independently selected from hydrogen or a C₁₋₃ aliphatic.Preferably, each R^(6b) is hydrogen or methyl. For compounds of formulaIII-A-aa in each of the above embodiments, the (S) stereochemistry ispreferred at the three position of the Ring A morpholine.

Examples of specific formula Y compounds are shown in Tables 3 and 4below.

TABLE 3 Specific examples of formula I compounds 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

Table 4 below shows specific examples of III-A-aa compounds.

TABLE 4 Specific examples of formula III-A-a compounds III-A-aa

No. R¹ R^(6b)/R^(6b) Q G 64 H CH₃/CH₃ CH₂

65 H CH₃/CH₃ CH₂

66 H CH₃/CH₃ CH₂

67 H CH₃/CH₃ CH₂ HO—CH₂CH₂N(CH₃)— 68 H CH₃/CH₃ CH₂

69 H CH₃/CH₃ CH₂

70 H CH₃/CH₃ CH₂

71 H CH₃/CH₃ CH₂

72 H CH₃/CH₃ CH₂ Et₂N— 73 H CH₃/CH₃ CH₂

74 H CH₃/CH₃ CH₂

75 H CH₃/CH₃ CH₂

76 H CH₃/CH₃ CH₂

77 H CH₃/CH₃ CH₂

78 H CH₃/CH₃ CH₂

79 H CH₃/CH₃ CH₂

80 H CH₃/CH₃ CH₂

81 CH₃ CH₃/CH₃ CH₂

82 H CH₃/CH₃ CH₂

83 H CH₃/CH₃ CH₂

84 CH₃ H/H CH₂

85 CH₃ H/H CH₂

86 H CH₃/CH₃ CH₂

87 CH₃ CH₃/CH₃ CH₂

88 CH₃ CH₃/CH₃ CH₂

89 F CH₃/CH₃ CH₂

90 F CH₃/CH₃ CH₂

91 H CH₃/CH₃ R—CH (CH₃)

92 H CH₃/CH₃ CH₂

93 H CH₃/CH₃ CH₂

94 H CH₃/CH₃ CH₂ Me₂N— 95 H CH₃/CH₃ CH₂ (CH₃O)(CH₃)N— 96 H CH₃/CH₃ CH₂

97 H CH₃/CH₃ CH₂

98 H CH₃/CH₃ CH₂

99 H CH₃/CH₃ CH₂

100 H CH₃/CH₃ CH₂

101 H CH₃/CH₃ CH₂

102 H CH₃/CH₃ CH₂

103 H CH₃/CH₃ CH₂

104 H CH₃/CH₃ CH₂

105 H CH₃/CH₃ CH₂

106 H CH₃/CH₃ CH₂

Based on their IκB kinase inhibitory properties and otherpharmacological properties, compound example numbers 1-30, 39-62 and64-106 are preferred. More preferred are compound example numbers 1, 2,7, 10, 11, 13, 16, 17, 19-27, 39-62, 64-106.

The compounds of the present invention may be administered to humans orother mammals by a variety of routes, including oral dosage forms andinjections (intravenous, intramuscular, intraperitoneal, subcutaneous,and the like). Numerous other dosage forms containing compounds of theinvention can be readily formulated by one skilled in the art, utilizingthe suitable pharmaceutical excipients (or carriers) as defined below.

Examples of pharmaceutically acceptable excipients (or carriers) andmethods of manufacture for various compositions can be found in A.Gennaro (ed.), Remington: The Science and Practice of Pharmacy, 20^(th)Edition, Lippincott Williams & Wilkins, Baltimore, Md. (2000), which isincorporated in its entirety by reference herein. Pharmaceuticallyacceptable excipients (or carriers) include flavoring agents,pharmaceutical-grade dyes or pigments, solvents, co-solvents, buffersystems, surfactants, preservatives, sweetener agents, viscosity agents,fillers, lubricants, glidants, disintegrants, binders and resins.

Conventional flavoring agents may be used, such as those described inRemington's Pharmaceutical Sciences, 18^(th) Ed., Mack Publishing Co.,pp. 1288-1300 (1990), which is incorporated in its entirety by referenceherein. The pharmaceutical compositions of the invention generallycontain from about 0 to 2%, of flavoring agents.

Conventional dyes and/or pigments may also be used, such as thosedescribed in the Handbook of Pharmaceutical Excipients, by the AmericanPharmaceutical Association & the Pharmaceutical Society of GreatBritain, pp. 81-90 (1986), which is incorporated in its entirety byreference herein. The pharmaceutical compositions of the inventiongenerally contain from about 0 to 2% of dyes and/or pigments.

The pharmaceutical compositions of the invention generally contain fromabout 0.1 to 99.9% of solvent(s). A preferred solvent is water.Preferred co-solvents include ethanol, glycerin, propylene glycol,polyethylene glycol, and the like. The pharmaceutical compositions ofthe invention may include from about 0 to 50% of co-solvents.

Preferred buffer systems include acetic, boric, carbonic, phosphoric,succinic, maleic, tartaric, citric, acetic, benzoic, lactic, glyceric,gluconic, glutaric and glutamic acids and their sodium, potassium andammonium salts. Particularly preferred buffers are phosphoric, tartaric,citric and acetic acids and salts thereof. The pharmaceuticalcompositions of the invention generally contain from about 0 to 5% of abuffer.

Preferred surfactants include polyoxyethylene sorbitan fatty acidesters, polyoxyethylene monoalkyl ethers, sucrose monoesters and lanolinesters and ethers, alkyl sulfate salts and sodium, potassium andammonium salts of fatty acids. The pharmaceutical compositions of theinvention generally contain from about 0 to 2% of surfactants.

Preferred preservatives include phenol, alkyl esters ofparahydroxybenzoic acid, o-phenylphenol benzoic acid and salts thereof,boric acid and salts thereof, sorbic acid and salts thereof,chlorobutanol, benzyl alcohol, thimerosal, phenylmercuric acetate andnitrate, nitromersol, benzalkonium chloride, cetylpyridinium chloride,methyl paraben and propyl paraben. Particularly preferred preservativesare the salts of benzoic acid, cetylpyridinium chloride, methyl parabenand propyl paraben. The pharmaceutical compositions of the inventiongenerally contain from about 0 to 2% of preservatives.

Preferred sweeteners include sucrose, glucose, saccharin, sorbitol,mannitol and aspartame. Particularly preferred sweeteners are sucroseand saccharin. Pharmaceutical compositions of the invention generallycontain from about 0 to 5% of sweeteners.

Preferred viscosity agents include methylcellulose, sodiumcarboxymethylcellulose, hydroxypropyl-methylcellulose,hydroxypropylcellulose, sodium alginate, carbomer, povidone, acacia,guar gum, xanthan gum and tragacanth. Particularly preferred viscosityagents are methylcellulose, carbomer, xanthan gum, guar gum, povidone,sodium carboxymethylcellulose, and magnesium aluminum silicate.Pharmaceutical compositions of the invention generally contain fromabout 0 to 5% of viscosity agents.

Preferred fillers include lactose, mannitol, sorbitol, tribasic calciumphosphate, dibasic calcium phosphate, compressible sugar, starch,calcium sulfate, dextro and microcrystalline cellulose. Pharmaceuticalcompositions of the invention generally contain from about 0 to 75% offillers.

Preferred lubricants/glidants include magnesium stearate, stearic acidand talc. Pharmaceutical compositions of the invention generally containfrom about 0 to 7%, preferably, about 1 to 5% of lubricants/glidants.

Preferred disintegrants include starch, sodium starch glycolate,crospovidone and croscarmelose sodium and microcrystalline cellulose.Pharmaceutical compositions of the invention generally contain fromabout 0 to 20%, preferably, about 4 to 15% of disintegrants.

Preferred binders include acacia, tragacanth, hydroxypropylcellulose,pregelatinized starch, gelatin, povidone, hydroxypropylcellulose,hydroxypropylmethylcellulose, methylcellulose, sugar solutions, such assucrose and sorbitol, and ethylcellulose. Pharmaceutical compositions ofthe invention generally contain from about 0 to 12%, preferably, about 1to 10% of binders.

Additional agents known to a skilled formulator may be combined with thecompounds of the invention to create a single dosage form.Alternatively, additional agents may be separately administered to amammal as part of a multiple dosage form.

For preparing pharmaceutical compositions containing the inventivecompounds, inert, pharmaceutically acceptable excipients or carriers canbe either solid or liquid. Solid form preparations include powders,tablets, dispersible granules, capsules, cachets and suppositories.

Generally, the powders and tablets may be comprised of from about 5 to95 weight percent of active ingredient. Suitable solid carriers areknown in the art, for example, magnesium carbonate, magnesium stearate,talc, sugar and lactose. Tablets, powders, cachets and capsules can beused as solid dosage forms suitable for oral administration. Examples ofpharmaceutically acceptable carriers and methods of manufacture forvarious compositions may be found in Remington's PharmaceuticalSciences, 18^(th) Ed., Mack Publishing Co. (1990).

Liquid form preparations include solutions, suspensions and emulsions.Common liquid form preparations include water and water-propylene glycolsolutions for parenteral injection or addition of sweeteners andopacifiers for oral solutions, suspensions and emulsions. Liquid formpreparations may also include solutions for intranasal administration.

Aerosol preparations suitable for inhalation include solutions andsolids in powder form, which may be combined with a pharmaceuticallyacceptable carrier, such as an inert compressed gas (e.g., nitrogen).

Also included are solid form preparations that may be converted, shortlybefore use, to liquid form preparations for either oral or parenteraladministration. Such liquid forms include solutions, suspensions andemulsions.

The compounds of the invention may also be delivered transdermally. Thetransdermal compositions can take the form of creams, lotions, aerosolsand emulsions and may be included in a transdermal patch of a matrix orreservoir type as is conventional in the art for this purpose.

The preferred mode of administering the compounds of the invention isoral. Preferably, the pharmaceutical preparation is in a unit dosageform. In such a form, the preparation is subdivided into suitable sizedunit doses containing appropriate quantities of the active component,for example, an effective amount to achieve the desired purpose.

The quantity of active ingredient (compound) in a unit dose ofpreparation may be varied or adjusted from about 0.01 to 4,000 mg,preferably, from about 0.01 to 1,000 mg, more preferably, from about0.01 to 500 mg, and even more preferably, from about 0.01 to 250 mg,according to the particular application. A typical recommended dailydosage regimen for oral administration will usually range from about0.02 to 2,000 mg/day, in one to four divided doses. For convenience, thetotal daily dosage may be divided and administered in portions duringthe day as required. Typically, pharmaceutical compositions of theinvention will be administered from about 1 to 5 times per day, oralternatively, as a continuous infusion. Such administration can be usedfor chronic or acute therapy. The amount of active ingredient that maybe combined with excipient or carrier materials to produce a singledosage form will vary depending upon the host treated and the particularmode of administration. A typical preparation will usually contain fromabout 5 to 95% of active compound (w/w). Preferably, such preparationswill contain from about 20 to 80 wt. % of active compound.

The pharmaceutically acceptable excipients or carriers employed inconjunction with the compounds of the invention are used at aconcentration sufficient to provide a practical size to dosagerelationship. The pharmaceutically acceptable excipients or carriers, intotal, can comprise from about 0.1 to 99.9% by weight of thepharmaceutical compositions of the invention, preferably, from about 20to 80% by weight.

Upon improvement of a patient's condition, a maintenance dose of acompound, composition or combination of the invention may beadministered, if applicable. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained. When thesymptoms have been alleviated to the desired level, treatment shouldcease. Patients may, however, require intermittent treatment on along-term basis upon any recurrence of disease symptoms.

Specific dosage and treatment regimens for any particular patient may bevaried and will depend upon a variety of factors, including the activityof the specific compound employed, the age, body weight, general healthstatus, sex and diet of the patient, the time of administration, therate of excretion, the specific drug combination, the severity andcourse of the symptoms being treated, the patient's disposition to thecondition being treated and the judgment of the treating physician.Determination of the proper dosage regimen for a particular situation iswithin the skill of the art. The amount and frequency of theadministration of compounds of the invention or their pharmaceuticallyacceptable salts may be regulated according to the judgment of theattending clinician, based on the factors recited above. As a skilledartisan will appreciate, lower or higher doses than those recited abovemay be required.

The inventive compounds are understood to provide efficacious treatmentof a variety of diseases, symptoms and disorders, particularly, thosewhich are inflammatory or immune-related in nature, including areasonable time of onset upon administration, and a reasonable durationafter administration. While food, diet, pre-existing conditions, alcoholand other systemic conditions could lengthen the time delay for aninventive drug to work after its administration, it is understood thatoptimum dosages will result in an efficacious drug treatment within andfor a reasonable amount of time.

The inventive compounds can exist in unsolvated as well as solvatedforms, including hydrated forms. In general, the solvated forms, withpharmaceutically acceptable solvents, such as water, ethanol and thelike, are equivalent to the unsolvated forms for purposes of thisinvention.

The inventive compounds may form pharmaceutically acceptable salts withorganic and inorganic acids. Examples of suitable acids for saltformation are hydrochloric, sulfuric, phosphoric, acetic, citric,malonic, salicylic, malic, fumaric, succinic, ascorbic, maleic,methanesulfonic and other mineral and carboxylic acids well known tothose skilled in the art. The salts are prepared by contacting the freebase forms with a sufficient amount of the desired acid to produce asalt in a conventional manner. The free base forms may be regenerated bytreating the salt with a suitable dilute aqueous base solution, such asdilute aqueous sodium hydroxide, potassium carbonate, ammonia or sodiumbicarbonate. The free base forms may differ somewhat from theirrespective salt forms in certain physical properties, such as solubilityin polar solvents, but the salts are otherwise equivalent to theirrespective free base forms for purposes of the invention,

On account of their pharmacological properties, the compounds accordingto the invention are suitable for the prophylaxis treatment and therapyof diseases, disorders and symptoms that involve increased activity ofIkB kinase. These include, for example, joint inflammation (e.g.,rheumatoid arthritis (RA), rheumatoid spondylitis, gouty arthritis,traumatic arthritis, rubella arthritis, psoriatic arthritis,osteoarthritis, and other arthritic conditions), acute synovitis,tuberculosis, atherosclerosis, muscle degeneration, cachexia, Reiter'ssyndrome, endotoxaemia, sepsis, septic shock, endotoxic shock, gramnegative sepsis, gout, toxic shock syndrome, pulmonary inflammatorydiseases (e.g., asthma, acute respiratory distress syndrome, chronicobstructive pulmonary disease, silicosis, pulmonary sarcoidosis, and thelike), bone resorption diseases, reperfusion injuries, carcinoses,leukemia, sarcomas, lymph node tumors, skin carcinoses, lymphoma,apoptosis, graft versus host reaction, graft versus host disease (GVHD),allograft rejection and leprosy.

Furthermore, the inventive compounds may be used in the treatment ofimmune-related diseases, symptoms and disorders, for example,infections, such as viral infections (e.g., HIV, cytomegalovirus (CMV),influenza, adenovirus, the Herpes group of viruses, and the like),parasitic infections (e.g., malaria, such as cerebral malaria), andyeast and fungal infections (e.g., fungal meningitis). In addition, theinventive compounds can be useful for treating fever and myalgias due toinfection, acquired immune deficiency syndrome (AIDS), AIDS relatedcomplex (ARC), cachexia secondary to infection or malignancy, cachexiasecondary to AIDS or cancer, keloid and scar tissue formation, pyresis,diabetes, and inflammatory bowel diseases (IBD) (e.g., Crohn's diseaseand ulcerative colitis). The compounds of the invention are also usefulin the treatment of diseases or injuries to the brain in whichover-expression of TNF-α has been implicated, such as multiple sclerosis(MS), ischemic brain injury, e.g. cerebral infarction (stroke) and headtrauma. The compounds of the invention are also useful in the treatmentof psoriasis, Alzheimer's disease, carcinomatous disorders (potentiationof cytotoxic therapies), cardiac infarct, chronic obstructive pulmonarydisease (COPD) and acute respiratory distress syndrome (ARDS).

In one embodiment the compounds of this invention are useful fortreating cancer, especially for treating cancers where IKK activity isabnormally high. The cancer types that may be treated include lymphoma,such as diffuse large B-cell, primary mediastinal B-cell, and mantlecell; multiple myeloma; osteolytic bone metastasis; head and necksquamous cell cancer; prostate cancer; pancreatic cancer and non-smallcell lung cancer. In one embodiment, the compounds are useful for ABClymphoma. For the treatment of cancer, the compounds may be used as asingle agent or in combination with other agents known to be useful forthe treatment of cancer. Examples of such other agents includebortezomib; capecitibine; gemcitabine; irinotecan; fludarabine;5-fluorouricil or 5-fluorouricil/leucovorin; taxanes, including, e.g.,paclitaxel and docetaxel; platinum agents, including, e.g., cisplatin,carboplatin, and oxaliplatin; anthracyclins, including, e.g.,doxorubicin and pegylated liposomal doxorubicin; mitoxantrone;dexamethasone; vincristine; etoposide; prednisone; thalidomide;Herceptin; temozolomide; and alkylating agents such as melphalan,chlorambucil, and cyclophosphamide.

The compounds of formula (I) are especially useful for treatinginflammatory and immune-related diseases, disorders and symptoms, moreespecially, inflammatory ones such as RA, asthma, IBD, psoriasis, COPDand MS. It will be appreciated that the present compounds are useful fortreating diseases, disorders or symptoms related to the activity ofNF-κB, TNF-α, and other enzymes in pathways where IKK is known tomodulate activity.

The compounds of this invention are also useful for treating a boneassociated disease, symptom or disorder in which there is a deficit ordeficiency of bone—either as a result of decreased new bone formation oran increase in bone resorption or a combination of both. Specificexamples include osteoporosis, periodontal disease, osteomyelitis,rheumatoid arthritis, aseptic joint loosening and osteolytic lesions(typically cancer related). It is known that rheumatoid arthritis, whichis characterized by inflammation of the joints, is also associated withdestruction of cartilage and bone. Furthermore, it has been reportedthat an IKK inhibitor provided inhibition of cartilage and bone loss ina murine model of collagen-induced arthritis. See McIntyre et al.,Arthritis & Rheumatism (2003), 48(9), 2652-2659.

Osteoporosis is a broad term applied to a number of distinct diseases inwhich there is decreased bone mass. These include primary osteoporosis(e.g., post-menopausal, senile osteoporosis and juvenile osteoporosis)and secondary osteoporosis. Examples of secondary osteoporosis would bethose associated with chronic diseases (e.g., chronic renal disease,hepatic insufficiency, gastrointestinal malabsorption, chronicimmobilization and chronic inflammatory diseases, including rheumatoidarthritis, osteoarthritis, periodontal disease and aseptic prostheticjoint loosening), endocrine dysfunction related diseases (e.g.,diabetes, hyperthyroidism, hyperparathyroidism, hypogonadism andhypopituitarism), drug and substance related symptoms (e.g.,corticosteroid, heparin, anticonvulsants, alcohol andimmunosupressants), and hematological disorders (e.g., metastaticdisease, myeloma, leukemia, gaucher's disease and anemia). Inhibition ofeither IkB directly or the NF-kB pathway indirectly has been reported tobe useful for the treatment of osteoporosis and osteoarthritis. See, forexample, PCT applications WO 2003104219, WO 2003103658, WO 2003029242,WO 2003065972, and WO 9965495. Accordingly, this invention also providesa method of treating or preventing bone loss in a patient in needthereof, comprising administering to the patient a compound of thisinvention. Also provided is a method of generating bone formation in apatient comprising administering a compound of this invention.

Another embodiment of the invention provides a method of inhibitingactivation of NF-κB dependent gene expression associated with theinhibition of IKK catalytic activity and/or IκB phosphorylation,comprising administering to a patient in need thereof an amount of thecompound according to claim 1 or the pharmaceutically acceptable salt orsolvate thereof, or a pharmaceutical composition thereof, which iseffective to inhibit IKK catalytic activity and/or IκB phosphorylation,thereby inhibiting activation of NF-κB dependent gene expression.

In one embodiment of the invention, there is provided a method oftreating or preventing an inflammatory or immune-related physiologicaldisorder, symptom or disease in a patient in need of such treatment,comprising administering to the patient an amount of at least onecompound according to claim 1, or the pharmaceutically acceptable saltor solvate thereof, or a pharmaceutical composition thereof, which iseffective to treat or prevent the inflammatory or immune-relatedphysiological disorder, symptom or disease. Preferably, the inflammatorydisease, disorder or symptom is rheumatoid arthritis, asthma, psoriasis,psoriatic arthritis, chronic obstructive pulmonary disease (COPD),inflammatory bowel disease or multiple sclerosis.

The invention comprises a compound having the formula (I), a method formaking an inventive compound, a method for making a pharmaceuticalcomposition from at least one inventive compound and at least onepharmaceutically acceptable carrier or excipient, and a method of usingone or more inventive compounds to treat a variety of disorders,symptoms and diseases, particularly ones that are inflammatory orimmune-related in nature. The inventive compounds and theirpharmaceutically acceptable salt and neutral compositions may beformulated together with a pharmaceutically acceptable excipient orcarrier and the resulting composition may be administered in vivo tomammals, such as primates, e.g. chimpanzees and humans (e.g. males andfemales) and animals (e.g., dogs, cats, cows, horses, and the like), totreat a variety of disorders, symptoms and diseases. Furthermore, theinventive compounds can be used to prepare a medicament that is usefulfor treating a variety of disorders, symptoms and diseases.

While one or more of the inventive compounds may be used in anapplication of monotherapy to treat a disorder, disease or symptom, theyalso may be used in combination therapy, in which the use of aninventive compound or composition (therapeutic agent) is combined withthe use of one or more other therapeutic agents for treating the sameand/or other types of disorders, symptoms and diseases. Combinationtherapy includes administration of the therapeutic agents concurrentlyor sequentially. Alternatively, the therapeutic agents can be combinedinto one composition which is administered to the patient.

In one embodiment, the compounds of this invention are used incombination with other therapeutic agents, such as other inhibitors ofIKK, other agents useful in treating NF-κB and TNF-α associatedconditions, and agents useful for treating other disorders, symptoms anddiseases. In particular, agents that induce apoptosis such as agentsthat disrupt cell cycle or mitochondrial function are useful incombination with the IKK inhibitors of this invention. Exemplary agentsfor combination with the IRK inhibitors include antiproliferative agents(e.g., methotrexate) and the agents disclosed in U.S. Pat. ApplicationPublication No. US2003/0022898, p 14, para. [0173-0174], which isincorporated herein in its entirety. In some embodiments, a compound ofthe invention is administered in conjunction with a therapeutic agentselected from the group consisting of cytotoxic agents, radiotherapy,and immunotherapy. Non-limiting examples of cytotoxic agents suitablefor use in combination with the IKK inhibitors of the invention includecapecitibine; gemcitabine; irinotecan; fludarabine; 5-fluorouracil or5-fluorouracil/leucovorin; taxanes, including, e.g., paclitaxel anddocetaxel; platinum agents, including, e.g., cisplatin, carboplatin, andoxaliplatin; anthracyclins, including, e.g., doxorubicin and pegylatedliposomal doxorubicin; mitoxantrone; dexamethasone; vincristine;etoposide; prednisone; thalidomide; herceptin; temozolomide; andalkylating agents such as melphalan, chlorambucil, and cyclophosphamide.It is understood that other combinations may be undertaken whileremaining within the scope of the invention.

Still another aspect of this invention is to provide a kit comprisingseparate containers in a single package, wherein the inventivepharmaceutical compounds, compositions and/or salts thereof are used incombination with pharmaceutically acceptable carriers to treatdisorders, symptoms and diseases where IkB kinase plays a role.

The compounds of this invention may be prepared by methods known tothose skilled in the art for analogous compounds, as illustrated by thegeneral schemes below, and by reference to the preparative examplesshown below.

Scheme I above shows a general route for obtaining compounds of formulaI. A Ring A carboxylic acid 1a may be coupled with the desired aminobeta-carboline 2a to provide 1. Many 1a intermediates that are usefulfor preparing compounds of this invention are readily available fromknown starting materials and chemical methods, especially in view of thesynthetic examples detailed herein. Schemes II-IV describe routes formaking the various β-carboline intermediates 2a.

Scheme II above shows a route for making a beta-carboline moiety whereR¹ is hydrogen, R² is chloro and R³ is alkoxy. While the scheme isexemplified for R³ being methoxy, it will be appreciated by one skilledin the art that beta-carbolines having other R³ alkoxy groups may beobtained by replacing NaOMe in step (e) with other sodium or metalalkoxides.

Scheme III above shows a route for preparing a beta-carbolineintermediate where R¹ is an alkyl such as methyl, R² is a halo such aschloro and R³ is hydrogen. One skilled in the art will understand howthe above scheme may be modified to obtain an R¹ alkyl group other thanmethyl or an R² halo group other than chloro.

Scheme IV above shows a route for making a beta-carboline intermediatewhere R¹ is fluoro, R² is chloro and R³ is hydrogen. It will beappreciated that ready modification of this scheme will allow for thepreparation of other intermediates. For example, another R² group may beintroduced by replacing the 4-chloro-2-iodoaniline in step (b) withanother 2-iodoaniline having a substituent other than chloro in the4-position.

A particularly useful intermediate for making compounds of formulaIII-A-aa is intermediate 3a:

where R¹³ is halo, OH, OR¹⁵, or a carboxylic acid protecting group; R¹⁴is an amino protecting group, hydrogen or —W-G as defined above; and R¹⁵is an organic radical. Amino protecting groups are well-known in theart. Examples of suitable amino protecting groups include alkoxycarbonylgroups such as t-butoxycarbonyl (t-BOC) and benzyl groups such as benzyland para-methoxybenzyl. The carboxylic acid group at the 3-position ofthe morpholine ring may be protected as any stable ester group such as asimple alkyl or aryl ester such as a methyl, ethyl, benzyl, orpentafluorophenyl ester. In one embodiment, R¹⁴ is —W-G and R¹³ is —OH,halo, or a carboxylic acid protecting group. Various protecting groupsare described in detail in Protecting Groups in Organic Synthesis,Theodora W. Greene and Peter G. M. Wuts, 3^(rd) edition, 1999, publishedby John Wiley and Sons.

A preferred enantiomer of intermediate 3a is (S)-3a:

where R¹³ and R¹⁴ are as described above.

Intermediate 3a or (S)-3a, as the carboxylic acid or an activated formthereof (such as the acid chloride), may be coupled with an appropriateamino-beta-carboline as outlined in Scheme I above. When R¹⁴ is an aminoprotecting group, the amide coupling reaction provides further usefulintermediates, shown below as compounds of formula IV:

where R¹⁴ is an amino protecting group and R¹, R² and R³ are asdescribed above. It will be appreciated by one of skill in the art thatcertain compounds of formula III-A-aa (where R^(6b) is each methyl) maybe prepared from compounds of formula IV by removing the R¹⁴ protectinggroup and then attaching the —W-G portion using known methods.Alternatively, the compounds of formula III-A-aa may be prepared byfirst constructing intermediate 3a where R¹⁴ is —W-G and R¹³ is acarboxylic acid or derivative thereof. The amide coupling reaction withan appropriate amino-beta-carboline then provides the compounds offormula III-A-aa directly.

Scheme V above shows a route for making intermediates of formula 3a,including the unprotected i-19. The selective protection anddeprotection of the amino and carboxylic acid groups in i-19 to providevarious 3a intermediates will be within the knowledge of one skilled inthe art.

Another useful intermediate for making compounds of formula III-A-aa isa compound of formula V, preferably (S)-V:

where R¹³ is halo or other leaving group, OH, OR¹⁵, or a carboxylic acidprotecting group, R¹⁵ is an organic radical such as a C₁₋₆ aliphatic,aryl or benzyl, Ring A has 0-2 or 0-4 R^(6b), and R¹, R², R³, and R^(6b)are as defined above.

Another useful intermediate for making compounds of this invention isVI, preferably (S)-VI:

where one of R¹³ and R^(13a) is OH or a leaving group such as halo andthe other is OR¹⁵ or a carboxylic acid protecting group, R¹⁵ is anorganic radical such as a C₁₋₆ aliphatic, aryl or benzyl, Ring A has0-2, and R^(6b) is as defined above.

SYNTHESIS EXAMPLES

The following abbreviations are used in the methods of preparation: RTor rt is room temperature; h, hr or hrs is hour or hours; min isminutes; TFA is trifluoroacetic acid; DMSO is dimethylsulfoxide; NCS isN-chlorosuccinimide; EDCI is1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; EtOAc isethyl acetate; DIEA is diisopropylethylamine; DCM is dichloromethane;DDQ is dichloro dicyano benzoquinone; mCPBA is meta-chloroperbenzoicacid; MeOH is methanol; EtOH is ethanol; MeCN is acetonitrile; TLC isthin layer chromatography; AIBN is azobisisobutyronitrile; NH₄OAc isammonium acetate; NaOAc is sodium acetate; Et₂O is diethyl ether; AcOHis acetic acid; and DMF is dimethylformamide. TBTU isN,N,N′,N′-tetramethyl-o-(benzotriazol-1-yl)uranium tetrafluoroborate.

INTERMEDIATE 1: 7-fluoro-2,3,4,9-tetrahydro-1H-β-carboline-1-carboxylicacid

To 10 g (46.6 mmol) of commercially available 6-fluorotryptaminehydrochloride was added 50 ml of 1M acetate buffer (pH 4.4) to give asuspension that was stirred at room temperature (RT). A suspension ofglyoxylic acid monohydrate (1.1 eq, 51.28 mmol, 4.72 g) in ethyl acetatewas then added to the stirred suspension in one portion. The suspensionwas stirred overnight (16 h) at RT and the precipitated solid wascollected by filtration and washed with both H₂O and ethyl acetate. Thesample was then dried in vacuo to give a light yellow solid inquantitative yield.

¹H-NMR (300 MHz, acetic acid-d₄): δ 3.04 (m, 2H), 3.56 (m, 1H), 3.83 (m,1H), 6.80 (m, 1H), 7.13 (dd, 1H), 7.34 (dd, 1H).

Retention Time (LC, method; ammonium acetate standard): 1.17 min.

MS (M+H⁺): 235.0.

INTERMEDIATE 2: 7-fluoro-2,3,4,9-tetrahydro-1H-β-carboline

7-fluoro-2,3,4,9-tetrahydro-1H-β-carboline-1-carboxylic acid (5 g, 21.36mmol) was suspended in 130 ml of 3N HCl in a 500 ml round-bottom flaskand refluxed overnight (16 hr) with stirring. Upon cooling, a lightbrown solid precipitated out, which was collected by filtration andwashed with H₂O. The salt obtained by filtration above was thendissolved in hot methanol (200 ml) and treated with 3M K₂CO₃ (5-10 ml)such that the pH is around 9. 100 ml of H₂O was added to this mixture,which was then allowed to stir at RT. The methanol was evaporated on arotary evaporator to give a white aqueous suspension of the desired freebase, which was collected by filtration (3.2 g, 79% yield).

¹H-NMR (300 MHz, methanol-d₄): δ 2.73 (t, 2H), 3.11 (t, 2H), 3.94 (s,2H), 6.73 (m, 1H), 6.94 (m, 1H), 7.30 (dd, 1H).

Retention Time (LC, method: ammonium acetate standard): 1.25 min.

MS (M+H⁺): 191.1.

INTERMEDIATE 3: 7-fluoro-9H-β-carboline

7-fluoro-2,3,4,9-tetrahydro-1H-β-carboline (3.5 g, 18.42 mmol) wassuspended in xylenes (60 ml) in a 250 ml round-bottom flask equippedwith a condenser that was open to the atmosphere, and heated. To thishot reaction mixture was added Pd/C (10 wt %, 0.2 eq, 700 mg) and themixture refluxed in xylenes overnight (12-14 hours). The solution wasthen filtered through a pad of celite and the collected filtrate wasthen evaporated on a rotary evaporator to give the desired product as abrown/tan solid (3.0 g, 88% yield).

¹H-NMR (300 MHz, DMSO-d₅): δ 7.10 (m, 1H), 7.37 (dd, 1H), 8.10 (d, 1H),8.28 (dd, 1H), 8.35 (dd, 1H), 8.89 (s, 1H), 11.74 (s, 1H).

Retention Time (LC, method: ammonium acetate standard): 1.88 min.

MS (M+H⁺): 187.1.

INTERMEDIATE 4: 6-chloro-7-fluoro-9H-β-carboline

7-fluoro-9H-β-carboline (2.15 g, 11.58 mmol) was suspended in 100 ml of1N HCl. To this mixture was added NCS (1.85 g, 13.89 mmol, 1.2 eq) andthe resulting mixture was stirred at RT overnight. The reaction mixturewas then filtered to give a light yellow solid (2.1 g, 83% yield).

¹H-NMR (300 MHz, DMSO-d₆): δ 7.86 (d, 1H), 8.64 (d, 1H), 8.79 (d, 1H),8.91 (d, 1H), 9.33 (s, 1H), 13.05 (s, 1H).

Retention Time (LC, method: ammonium acetate standard): 2.19 min.

MS (M+H⁺): 221.1.

INTERMEDIATE 5: 6-chloro-7-fluoro-8-nitro-9H-β-carboline

6-chloro-7-fluoro-9H-β-carboline (2.1 g, 9.54 mmol) was taken in around-bottom flask (250 ml) and NaNO₃ (1.136 g, 13.36 mmol, 1.4 eq) wasadded. TFA (48 ml) was then added to the flask and the resulting mixturerefluxed overnight. The TFA is then removed on a rotary evaporator. Theresulting slurry is suspended in water (50 ml) and sonicated thoroughly.The resulting suspension is then filtered to give a yellow solid (2.0 g,80% yield).

¹H-NMR (300 MHz, DMSO-d₆): δ 8.21 (d, 1H), 8.46 (d, 1H), 9.04 (m, 2H),12.55 (s, 1H).

Retention Time (LC, method: ammonium acetate standard): 2.24 min.

MS (M+H⁺): 266.2.

INTERMEDIATE 6: 6-chloro-7-methoxy-8-nitro-9H-β-carboline

Methanol (0.462 ml, 11.4 mmol) was added to a stirring suspension of NaH(684 mg, 17.1 mmol) in DMF (10 ml) under an argon atmosphere. Theresulting solution was allowed to stir at RT for 20 min.6-chloro-7-fluoro-8-nitro-9H-β-carboline (500 mg, 1.9 mmol) was added tothe stirring solution and the resulting mixture was allowed to stir atRT. Upon addition of H₂O, a brown solid precipitated out which wasfiltered to give the desired 6-chloro-7-methoxy-8-nitro-9H-β-carboline(510 mg, 97% yield).

¹H-NMR (300 MHz, DMSO-d₆): δ 4.02 (s, 3H), 8.52 (d, 1H), 8.60 (d, 1H),9.05 (s, 1H), 9.12 (s, 1H), 12.78 (b, 1H).

Retention Time (LC, method: ammonium acetate standard): 2.28 min.

MS (M+H⁺): 278.

INTERMEDIATE 7: 6-chloro-7-methoxy-9H-β-carboline-8-ylamine

6-chloro-7-methoxy-8-nitro-9H-β-carboline (510 mg, 1.84 mmol) wassuspended in 50 ml of methanol and 100 mg of Pd/C (10%) was added. Theflask was fitted with a balloon of hydrogen and the reaction mixture wasstirred overnight at RT. Upon filtration through a pad of celite andevaporation of the methanol, a dark brown solid was obtained. Thisresidue was suspended in methanol (15 ml) and added, with vigorousstirring, to a solution of saturated NaHCO₃ (100 ml). The light brownsolid that precipitated out was collected by filtration and driedthoroughly in vacuo to give the desired product (512 mg, quantitativeyield).

¹H-NMR (300 MHz, methanol-d₄): δ 3.90 (s, 3H), 7.63 (s, 1H), 8.11 (d,1H), 8.27 (d, 1H), 8.84 (s, 1H).

Retention Time (LC, method: ammonium acetate standard): 1.12 min.

MS (M+H⁺): 248.

INTERMEDIATE 8: 6-chloro-9H-β-carboline-8-ylamine

The target compound was prepared according to the procedures outlinedfor Intermediate 1 to Intermediate 7 where the starting material forIntermediate 1 was unsubstituted tryptamine. An alternative synthesisfor 6-chloro-9H-β-carboline-8-ylamine is described on page 34, example15 of PCT Application Publication No. WO 01/68648 A1, which isincorporated herein in its entirety.

METHOD A: COUPLING PROCEDURE FOR 6,7,8-SUBSTITUTED-8-CARBOLINES

6,7,8-substituted-9H-β-carboline (1 mmol), EDCI (1.6 mmol) and theappropriate carboxylic acid (1.2 mmol) were taken in a round-bottomflask and suspended in pyridine (5 ml). The resulting mixture was heatedat 60° C. overnight. The pyridine was then removed by rotary evaporationand 5% Na₂CO₃ solution was added. The resulting solid that precipitatedout was collected by filtration. Chromatographic purification gave thedesired product.

METHOD B: COUPLING PROCEDURE FOR 6,8-SUBSTITUTED-β-CARBOLINES

6,8-substituted-9H-β-carboline (1.0 mmol), EDCI (1.6 mmol) and thecarboxylic acid (1.2 mmol) to be coupled were taken in a round-bottomflask and suspended in pyridine (5 ml). The resulting mixture wasstirred overnight. The pyridine was then removed by rotary evaporationand 5% Na₂CO₃ solution was added. The resulting solid that precipitatedout was collected by filtration. Chromatographic purification gave thedesired product.

Example 1N-(6-chloro-7-methoxy-9H-β-carbolin-8-yl)-2-methyl-nicotinamide

6-chloro-7-methoxy-9H-β-carbolin-8-ylamine (100 mg, 0.4 mmol), EDCI (125mg, 0.64 mmol) and 2-methyl nicotinic acid (66 mg, 0.48 mmol) were takenin a round-bottom flask and suspended in pyridine (2 ml). The resultingmixture was heated at 80° C. overnight. The pyridine was then removed byrotary evaporation and 5% Na₂CO₃ solution was added. The resulting solidthat precipitated out was collected by filtration, Chromatographicpurification gave the desired product in 50-70% yield.

¹H-NMR (300 MHz, DMSO-d₆): δ 2.71 (s, 3H), 3.89 (s, 3H), 7.45 (dd, 1H),8.15 (d, 1H), 8.21 (d, 1H), 8.38 (d, 1H), 8.45 (s, 1H), 8.61 (d, 1H),8.92 (s, 1H), 10.33 (s, 1H), 11.57 (s, 1H).

Retention Time (LC, method: ammonium acetate standard): 1.77 min.

MS (M+H⁺): 367.1.

Example 2 4-methyl-pyrimidine-5-carboxylic acid(6-chloro-7-methoxy-9H-beta-carbolin-8-yl)-amide

The desired compound was prepared according to Method A from6-chloro-7-methoxy-9H-beta-carbolin-8-ylamine and 4-methyl-5-pyrimidinecarboxylic acid in 80% yield.

¹H-NMR (300 MHz, DMSO-d₆): δ 2.75 (s, 3H), 3.92 (s, 3H), 8.19 (d, 1H),8.41 (d, 1H), 8.50 (s, 1H), 8.96 (s, 1H), 9.20 (s, 1H), 9.25 (s, 1H),10.63 (s, 1H), 11.67 (s, 1H).

Retention Time (LC, method: formic acid standard): 0.95 min.

MS (M+H⁺): 368.

INTERMEDIATE 9: 6-chloro-7-ethoxy-8-nitro-9H-β-carboline

Sodium ethoxide (232 mg, 3.4 mmol) was added to a solution of6-chloro-7-fluoro-8-nitro-9H-β-carboline (200 mg, 0.76 mmol) in DMSO (4ml) and the reaction mixture allowed to stir overnight. The reactionmixture was diluted with water and the pH of the solution was adjustedto about 4 by adding 1N HCl. The aqueous solution was extracted (3×)with EtOAc. The combined EtOAc layers were dried and evaporated. Thecrude product was purified by flash chromatography to give the desiredproduct in 40-60% yield.

¹H-NMR (300 MHz, DMSO-d₅): δ 1.44 (t, 3H), 4.24 (q, 2H), 8.21 (d, 1H),8.46 (d, 1H), 8.91 (s, 1H), 9.02 (s, 1H).

Retention Time (LC, method: ammonium acetate standard): 2.37 min.

MS (M+H⁺): 291.9.

INTERMEDIATE 10: 6-chloro-7-ethoxy-9H-β-carboline-8-ylamine

6-chloro-7-ethoxy-8-nitro-9H-β-carboline (160 mg, 0.55 mmol) wassuspended in 4 ml of methanol and 25 mg of Pd/C (10%) was added. Theflask was fitted with a balloon of hydrogen and the reaction mixture wasstirred overnight at RT. Upon filtration through a pad of celite andevaporation of the methanol, a dark brown solid was obtained anddetermined to be the desired6-chloro-7-cyclopropylmethoxy-9H-β-carbolin-8-ylamine (80 mg, 55%).

¹H-NMR (300 MHz, Methanol-d₄/CDCl₃): ±1.26 (t, 3H), 3.91 (q, 2H), 7.34(s, 1H), 7.71 (d, 1H), 8.02 (s, 1H), 8.56 (s, 1H).

Retention Time (LC, method: formic acid standard): 1.16 min.

MS (M+H⁺): 262.0.

Example 3 N-(6-chloro-7-ethoxy-9H-β-carbolin-8-yl)-2-methyl-nicotinamide

The desired compound was prepared according to Method A from6-chloro-7-ethoxy-9H-β-carbolin-8-ylamine and 2-methylnicotinic acid in40% yield.

¹H-NMR (300 MHz, MeOH-d₄): β 1.39 (t, 3H), 2.76 (s, 3H), 4.14 (q, 2H),7.43 (dd, 1H), 8.05 (d, 1H), 8.26 (m, 3H), 8.55 (d, 1H), 8.79 (s, 1H).

Retention Time (LC, method: ammonium acetate standard): 1.98 min.

MS (M+H⁺): 381.3.

INTERMEDIATE 11: 6-chloro-7-(N,N)-dimethylamino-8-nitro-9H-β-carboline

N,N-dimethylamine hydrochloride (278 mg, 3.4 mmol) was added to astirring solution of 6-chloro-7-fluoro-8-nitro-9H-β-carboline (300 mg,1.13 mmol) in DMSO (8 ml) under an argon atmosphere. This was followedby the addition of DIEA (0.83 ml, 4.65 mmol) and the reaction mixturewas heated at 60° C. overnight. After allowing the reaction mixture tocool to RT, water was added and a dark orange solid precipitated out.The solid was filtered, washed with water and dried to give the desiredproduct (230 mg, 70% yield).

¹H-NMR (300 MHz, DMSO-d₆): δ 4.06 (s, 6H), 7.23 (d, 1H), 7.53 (d, 1H),7.66 (s, 1H), 8.05 (s, 1H),

Retention Time (LC, method: ammonium acetate standard): 2.41 min.

MS (M+H⁺): 290.9.

INTERMEDIATE 12: 6-chloro-7-(N,N)-dimethylamino-9H-β-carboline-8-ylamine

6-chloro-7-(N,N)-dimethylamino-8-nitro-9H-β-carboline (828 mg, 2.86mmol) was suspended in 30 ml of methanol and 166 mg of Pd/C (10%) wasadded. The flask was fitted with a balloon of hydrogen and the reactionmixture was stirred overnight at ambient temperature. Upon filtrationthrough a pad of celite and evaporation of the methanol, a dark brownsolid was obtained and determined to be the desired6-chloro-7-(N,N)-dimethylamino-9H-β-carboline-8-ylamine (500 mg, 67%yield).

¹H-NMR (300 MHz, DMSO-d₆): δ 2.80 (s, 6H), 5.42 (s, 2H), 7.45 (s, 1H),7.95 (d, 1H), 8.23 (d, 1H), 8.86 (d, 1H).

Retention Time (LC, method: ammonium acetate standard): 2.32 min.

MS (M+H⁺): 261.1.

Example 4N-(6-chloro-7-7-(N,N)-dimethylamino-9H-β-carbolin-8-yl)-2-methyl-nicotinamide

The desired compound was prepared according to Method A from6-chloro-7-(N,N)-dimethylamino-9H-β-carboline-8-ylamine and2-methylnicotinic acid in 40-60% yield.

¹H-NMR (300 MHz, methanol-d₄/CDCl₃): δ 2.82 (s, 3H), 2.94 (s, 6H), 7.33(m, 1H), 8.02 (m, 3H), 8.34 (d, 1H), 8.61 (d, 1H), 8.95 (s, 1H).

Retention Time (LC, method: ammonium acetate standard): 2.29 min.

MS (M+H⁺): 380.3.

INTERMEDIATE 13:6-chloro-7-(4-methyl-piperazin-1-yl)-8-nitro-9H-β-carboline

To a DMSO solution (4 ml) of 200 mg (0.755 mmol) of6-chloro-7-fluoro-8-nitro-9H-β-carboline was added 1-methylpiperazine(226 mg, 2.26 mmol) and DIEA (400 mg, 3.09 mmol) via a syringe. Thereaction was allowed to stir at RT overnight. Upon addition of water, anorange solid precipitated out. The solid was filtered, washed and driedto give 236 mg (91% yield) of the desired6-chloro-7-(4-methyl-piperazin-1-yl)-8-nitro-9H-β-carboline.

¹H-NMR (300 MHz, DMSO-d₆): δ 2.24 (s, 3H), 2.48 (m, 4H), 3.13 (m, 4H),8.13 (d, 1H), 8.40 (d, 1H), 8.73 (s, 1H), 8.94 (s, 1H), 12.05 (s, 1H).

Retention Time (LC, method: ammonium acetate standard): 1.72 min.

MS (M+H⁺): 346.

INTERMEDIATE 14:6-chloro-7-(4-methyl-piperazin-1-yl)-8-amino-9H-β-carboline

Suspended 236 mg of6-chloro-7-(4-methyl-piperazin-1-yl)-8-nitro-9H-β-carboline in 100 ml ofmethanol and added 10% Pd/C (48 mg) under argon. The flask was flushedwith hydrogen (3×) and the reaction mixture was stirred under a hydrogenatmosphere at RT overnight. The reaction mixture was filtered and Pd/Cwas removed using a pad of celite. The reaction mixture was evaporatedto remove solvent and purified by flash chromatography to give 119 mg(55% yield) of the desired6-chloro-7-(4-methyl-piperazin-1-yl)-8-amino-9H-β-carboline.

¹H-NMR (300 MHz, methanol-d₄): δ 2.39 (s, 3H), 2.46 (m, 2H), 2.87 (m,4H), 3.83 (m, 2H), 7.50 (s, 1H), 7.97 (d, 1H), 8.24 (d, 1H), 8.78 (s,1H).

Retention Time (LC, method: ammonium acetate polar): 1.32 min.

MS (M+H⁺): 316.

Example 52-chloro-N-[6-chloro-7-(4-methyl-piperazin-1-yl)-9H-β-carbolin-8-yl]-nicotinamide

The desired compound was prepared according to Method A from6-chloro-7-(4-methyl-piperazin-1-yl)-8-amino-9H-β-carboline and2-chloro-nicotinic acid in 25% yield.

¹H-NMR (300 MHz, DMSO-d₆): δ 2.21 (s, 3H), 2.33 (m, 2H), 2.54 (m, 2H),3.24 (m, 4H), 7.73 (dd, 1H), 8.12 (d, 1H), 8.35 (d, 1H), 8.37 (s, 1H),8.48 (dd, 1H), 8.61 (dd, 1H), 8.91 (s, 1H), 10.40 (s, 1H), 11.33 (s,1H).

Retention Time (LC, method: ammonium acetate standard): 1.46 min.

MS (M+H⁺): 455.

INTERMEDIATE 15: Resolution of rac-terebic acid

Terebic acid was dissolved in a 10:1 mixture of EtOAc-MeOH [10 g ofterebic acid (17.7 g of salt) in 550 ml] and heated to 50-55° C.,followed by addition of (S)-(−)-α-methyl-benzylamine. The reactionmixture was stirred for 2 minutes and then left at RT for 15 minutes.The reaction mixture was then seeded with enriched salt (prepared on asmaller scale using 3 recrystallization cycles), sonicated for 10-15seconds and left at RT overnight. The solid was filtered off, washedwith EtOAc and dried under vacuum. Recrystallization was done in thesame solvent mixture by re-dissolving the salt (24 mg/ml). This mixturewas then heated to a gentle reflux for a short period of time (fewcrystals remained in suspension). The mixture was left at RT over night.The solid was processed as previously described.

Enantiomeric excess was determined in a crude fashion using proton NMRof the corresponding amide obtained from TBTU coupling.

Regeneration of (R)-(+)-terebic acid: The salt was dissolved in water(320 mg/ml), heated to 65° C. and 1.2 equivalent of aqueous 6M HCl wasadded. The reaction mixture was then left at 4° C. overnight. The solidwas filtered off, washed with small portions of cold water and driedunder a high vacuum (yield of 25-30%).

¹H-NMR (300 MHz, DMSO-d₆): δ 1.30 (s, 3H), 1.52 (s, 3H), 2.74 (dd, 1H),2.85 (dd, 1H), 3.25 (t, 1H).

Retention Time (LC, method: ammonium acetate standard): 0.33 min.

MS (M+H⁺): 159.0.

Example 6 2,2-dimethyl-5-oxo-tetrahydro-furan-3-carboxylic acid(6-chloro-9H-β-carbolin-8-yl)-amide

The desired compound was prepared according to Method B from6-chloro-9H-β-carboline-8-ylamine and (R)-terebic acid (Intermediate 15)in 80-90% yield.

¹H-NMR (300 MHz, DMSO-d₆): δ 1.44 (s, 3H), 1.59 (S, 3H), 2.91 (dd, 1H),3.08 (dd, 1H), 3.38 (dd, 1H), 7.85 (m, 1H), 8.17 (d, 1H), 8.25 (d, 1H),8.39 (d, 1H), 9.05 (s, 1H), 10.26 (s, 1H), 11.72 (s, 1H).

Retention Time (LC, method: ammonium acetate standard): 1.22 min.

MS (M+H⁺): 358.3.

Example 7 1,2,2-trimethyl-5-oxo-pyrrolidine-3-carboxylic acid(6-chloro-9H-β-carbolin-8-yl)-amide

The desired compound was prepared according to Method B from6-chloro-9H-β-carboline-8-ylamine and1,2,2-trimethyl-5-oxo-pyrrolidine-3-carboxylic acid in 68% yield.

¹H-NMR (DMSO-d₆, 300 MHz) δ 1.23 (s, 3H), 1.45 (s, 3H), 2.59 (dd, 1H),2.66 (s, 3H), 2.70 (dd, 1H), 3.17 (t, 1H), 7.91 (m, 1H), 8.18 (d, 1H),8.24 (d, 1H), 8.40 (d, 1H), 9.07 (s, 1H), 10.16 (s, 1H), 11.32 (s, 1H).

Retention Time (LC, method: ammonium acetate standard): 1.17 min.

MS (M+H⁺): 371.3.

INTERMEDIATE 16: N-benzyl-serine benzyl ester

To a mixture of L-serine-benzyl ester-HCl (2.3 g), benzaldehyde (1.05eq.) and sodium acetate (1 eq.) in methanol was added sodiumcyanoborohydride (1.0 eq.). The resulting mixture was stirred at ambienttemperature for 15 hrs, then partitioned into ether and aqueoussaturated sodium bicarbonate. The separated organic phase was extractedwith 1M HCl (3×). Combined aqueous extracts were washed with ether,basified with aqueous 4.5M K₂CO₃ and extracted with ether. Combinedorganic extracts were washed with brine, dried over sodium sulfate andconcentrated to dryness to give 2.32 g of the desired compound (waxysolid, 81% yield).

¹H-NMR (300 MHz, CDCl₃): δ 3.48 (dd, 1H), 3.64 (dd, 1H), 3.74 (d, 1H),3.80 (dd, 1H), 3.88 (d, 1H), 5.18 (s, 2H), 7.25-7.39 (m, 10H).

MS (M+H⁺): 286.

INTERMEDIATE 17: 4-benzyl-6-iodomethyl-6-methyl-morpholine-3-carboxylicacid benzyl ester

To a solution of N-benzyl-serine benzyl ester (Intermediate 16, 6.35 g)in 90 ml of MeCN at ambient temperature was added3-bromo-2-methyl-propene (5.6 ml), KI (0.740 mg) and K₂CO₃ (7.7 g). Thereaction mixture was stirred at ambient temperature for 72 hrs. 1 ml of3-bromo-2-methyl-propene was added and the reaction mixture was stirredfor another 15 hrs. Only a small amount of starting material remainedbased on TLC (1:1; EtOAc-hexane). To the resulting mixture was added11.2 g of iodine. After 4 hrs of stirring, TLC (10% EtOAc/hexane) showedcomplete conversion. The reaction mixture was partitioned into ether(300 ml) and 0.5 M Na₂S₂O₃ (100 ml). The separated organic phase waswashed successively with water, saturated NaHCO₃ and brine, dried overMgSO₄, and concentrated to dryness. The residue was purified on silica(5% EtOAc/Hexane) to give 6.65 g (yellowish oil, 64% yield, about 4:1mixture) of compound4-benzyl-6-iodomethyl-6-methyl-morpholine-3-carboxylic acid benzylester. Major Component: ¹H-NMR (300 MHz, CDCl₃): δ 1.22 (s, 3H), 2.50(d, 1H), 3.16-3.36 (m, 3H), 3.75-3.95 (m, 4H), 4.06 (dd, 1H), 5.16 (d,1H), 5.21 (d, 1H), 7.28-7.36 (m, 10H).

MS (M+H⁺): 466.

INTERMEDIATE 18: 4-benzyl-6,6-dimethyl-morpholine-3-carboxylic acidbenzyl ester

To a solution of 4-benzyl-6-iodomethyl-6-methyl-morpholine-3-carboxylicacid benzyl ester (Intermediate 17, 1.23 g) and tributyltin hydride (1.8ml, 2.5 eq.) in 11 ml of toluene under gentle reflux was added over 1.5hr a solution of AIBN in toluene (25 mg/l ml). The mixture was allowedto cool down and was concentrated to dryness. The residue waspartitioned into 15% 1M HCl in acetonitrile and hexane. The separatedhexane phase was extracted two times with the acetonitrile solution. Thecombined acetonitrile solutions were washed with hexane two times andconcentrated. The residue was partitioned into ether and 1M K₂CO₃. Theseparated ether phase was washed successively with 0.4M Na₂S₂O₃ andbrine, dried over MgSO₄ and concentrated. The residue was purified onsilica (7.5% EtOAc/hexane) to give 760 mg (oil, 85% yield) of compound4-benzyl-6,6-dimethyl-morpholine-3-carboxylic acid benzyl ester.

¹H-NMR (300 MHz, CDCl₃): δ 1.21 (s, 3H), 1.24 (s, 3H), 2.12 (d, 1H),2.84 (d, 1H), 3.29 (t, 1H), 3.58 (d, 1H), 3.95-4.05 (m, 2H), 5.15 (d,1H), 5.21 (d, 1H), 7.28-7.36 (m, 10H).

MS (M+H⁺): 340.

INTERMEDIATE 19: 6,6-dimethyl-morpholine-3-carboxylic acid

To a solution of 4-benzyl-6,6-dimethyl-morpholine-3-carboxylic acidbenzyl ester (Intermediate 18, 1.25 g) in 40 ml of 10% AcOH/MeOH (undernitrogen) was added 250 mg of 20% Pd(OH)₂ on charcoal. The reactionmixture was purged with hydrogen (balloon) and was stirred at ambienttemperature for 72 hrs. To the resulting gray mixture was added 4 ml ofwater to help dissolution. The catalyst was removed by filtration andthe filtrate was concentrated to dryness. The residue was co-evaporatedwith ethanol (2×) and then triturated with EtOAc. The generated whitesolid was filtered off and dried under high vacuum to give 559 mg of6,6-dimethyl-morpholine-3-carboxylic acid (95% yield).

¹H-NMR (300 MHz, D₂O): δ 1.35 (s, 3H), 1.38 (s, 3H), 3.11 (d, 1H), 3.32(d, 1H), 3.81-3.87 (m, 1H), 4.05 (bt, 1H), 4.17 (bd, 1H).

MS (M+H⁺): 160.

INTERMEDIATE 20: 4,6,6-trimethyl-morpholine-3-carboxylic acid

To a suspension of 6,6-dimethyl-morpholine-3-carboxylic acid(Intermediate 19, 540 mg) in 17 ml of ethanol (under nitrogen) was added100 mg of 10% Pd on charcoal and 830 ul (˜3 eq.) of 37% aqueousformaldehyde. The mixture was purged with hydrogen (balloon) and stirredat ambient temperature for 5 hrs. To the resulting gray mixture wereadded 4 ml of water and 4 ml of methanol to help dissolution. Thecatalyst was removed by filtration and the filtrate was concentrated todryness. The residue was triturated with EtOAc. The generated whitesolid was filtered off and dried under high vacuum to give 574 mg of4,6,6-trimethyl-morpholine-3-carboxylic acid (97% yield).

¹H-NMR (300 MHz, D₂O): δ 1.35 (s, 3H), 1.43 (s, 3H), 2.94 (s, 3H), 3.08(d, 1H), 3.41 (d, 1H), 3.68 (dd, 1H), 3.96 (t, 1H), 4.14 (dd, 1H).

MS (M+H⁺): 174.

Example 8 4,6,6-trimethyl-morpholine-3-carboxylic acid(6-chloro-9H-β-carbolin-8-yl)-amide

The desired compound was prepared according to Method B from6-chloro-9H-β-carboline-8-ylamine and4,6,6-trimethyl-morpholine-3-carboxylic acid in 61% yield.

¹H-NMR (DMSO-d₆, 300 MHz) δ 1.15 (s, 3H), 1.39 (s, 3H), 1.98 (d, 1H),2.26 (s, 3H), 2.72 (d, 1H), 2.80 (dd, 1H), 3.79 (m, 2H), 7.91 (s, 1H),8.03-8.08 (m, 2H), 8.22 (d, 1H), 8.97 (s, 1H).

Retention Time (LC, method: ammonium acetate standard): 1.33 min.

MS (M+H⁺): 373.2.

Example 9 2,2-Dimethyl-5-oxo-tetrahydro-furan-3-carboxylic acid(6-chloro-7-methoxy-9H-β-carbolin-8-yl)-amide

The desired compound was prepared according to Method A from6-chloro-7-methoxy-9H-β-carboline-8-ylamine and (R)-terebic acid in a60-80% yield.

¹H-NMR (300 MHz, DMSO-d₆): δ 1.48 (s, 3H), 1.61 (s, 3H), 3.03 (m, 2H),3.51 (m, 1H), 3.86 (s, 3H), 8.16 (m, 1H), 8.37 (m, 1H), 8.43 (s, 1H),8.94 (s, 1H), 10.10 (s, 1H), 11.33 (s, 1H).

Retention Time (LC, method: ammonium acetate standard): 1.94 min.

MS (M+H⁺): 388.

INTERMEDIATE 21: 6-chloro-7-cyclopropylmethoxy-8-nitro-9H-β-carboline

Cyclopropylmethyl alcohol (0.921 ml, 11.4 mmol) was added to a stirringsuspension of NaH (455 mg, 11.4 mmol) in DMF (20 ml) under an argonatmosphere. The resulting solution was allowed to stir at RT for 20 min.6-chloro-7-fluoro-8-nitro-9H-β-carboline (500 mg, 1.9 mmol) was added tothe stirring solution and the resulting mixture was allowed to stir atRT. Upon addition of H₂O, a brown solid precipitated out which wasfiltered to give the desired6-chloro-7-cyclopropylmethyoxy-8-nitro-9H-β-carboline (510 mg, 85%).

¹H-NMR (300 MHz, DMSO-d₆): δ 0.35 (m, 2H), 0.59 (m, 2H), 1.32 (m, 1H),4.04 (d, 2H), 8.21 (d, 1H), 8.46 (d, 1H), 8.90 (s, 1H), 9.02 (s, 1H),12.32 (b, 1H)

Retention Time (LC, method: ammonium acetate standard): 2.63 min.

MS (M+H⁺): 318.

INTERMEDIATE 22: 6-chloro-7-cyclopropylmethoxy-9H-β-carbolin-8-ylamine

6-chloro-7-cyclopropylmethoxy-8-nitro-9H-β-carboline (510 mg, 1.61 mmol)was suspended in 12 ml of methanol and 100 mg of Pd/C (10%) was added.The flask was fitted with a balloon of hydrogen and the reaction mixturewas stirred overnight at RT. Upon filtration through a pad of celite andevaporation of the methanol, a dark brown solid was obtained. Thisresidue was suspended in methanol (10 ml) and added, with vigorousstirring, to a solution of saturated NaHCO₃ (100 ml). The light brownsolid that precipitated out was collected by filtration and driedthoroughly in vacuo to give the desired6-chloro-7-cyclopropylmethoxy-9H-β-carbolin-8-ylamine (371 mgs, 80%yield).

¹H-NMR (300 MHz, methanol-d₄): δ 0.36 (m, 2H), 0.61 (m, 2H), 1.37 (m,1H), 3.88 (d, 2H), 7.58 (s, 1H), 7.96 (d, 1H), 8.23 (d, 1H), 8.76 (s,1H).

Retention Time (LC, method: ammonium acetate standard): 2.28 min.

MS (M+H⁺): 288.

Example 10N-(6-chloro-7-cyclopropylmethoxy-9H-β-carbolin-8-yl)-2-methyl-nicotinamide

The desired compound was prepared according to Method A from6-chloro-7-cyclopropylmethoxy-9H-β-carbolin-8-ylamine and2-methylnicotinic acid in a 40-60% yield.

¹H-NMR (300 MHz, MeOH-d₄): δ 0.29 (m, 2H), 0.55 (m, 2H), 1.29 (m, 1H),2.78 (s, 3H), 3.95 (m, 2H), 7.46 (dd, 1H), 8.08 (m, 1H), 8.30 (m, 3H),8.59 (m, 1H), 8.81 (s, 1H).

Retention Time (LC, method: ammonium acetate standard): 2.18 min.

MS (M+H⁺): 405.

INTERMEDIATE 23:6-chloro-7-(N,N)-dimethylaminoethoxy-8-nitro-9H-β-carboline

N,N-dimethylaminoethyl alcohol (6.0 eq) was added to a stirringsuspension of NaH (6.0 eq) in DMF under an argon atmosphere. Theresulting solution was allowed to stir at RT for 20 min.6-chloro-7-fluoro-8-nitro-9H-β-carboline (1.0 eq) was added to thestirring solution and the resulting mixture was allowed to stir at RT.Upon addition of H₂O, a brown solid precipitated out which was filteredto give the desired6-chloro-7-(N,N)-dimethylaminoethoxy-8-nitro-9H-□-carboline(quantitative yield).

¹H-NMR (300 MHz, DMSO-d₆): δ 2.23 (s, 6H), 2.74 (t, 2H), 4.28 (t, 2H),8.21 (d, 1H), 8.46 (d, 1H), 8.90 (s, 1H), 9.02 (s, 1H).

Retention Time (LC, method: ammonium acetate standard): 1.26 min.

MS (M+H⁺): 335.

INTERMEDIATE 24:6-chloro-7-(N,N)-dimethylaminoethoxy-9H-β-carbolin-8-ylamine

6-chloro-7-(N,N)-dimethylaminoethoxy-8-nitro-9H-β-carboline (500 mg, 1.5mmol) was suspended in 12 ml of methanol and 100 mg of Pd/C (10%) wasadded. The flask was fitted with a balloon of hydrogen and the reactionmixture was stirred overnight at RT. Upon filtration through a pad ofcelite and evaporation of the methanol, a dark brown solid was obtained.The residue was suspended in methanol (10 ml) and added, with vigorousstirring, to a solution of saturated NaHCO₃ (100 ml). The solid thatprecipitated out was collected by filtration and dried thoroughly invacuo to give the desired6-chloro-7-(N,N)-dimethylaminoethoxy-9H-β-carboline-8-ylamine (380 mgs,83%).

¹H-NMR (300 MHz, methanol-d₄): δ 2.43 (s, 6H), 2.84 (t, 2H), 4.11 (t,2H), 7.47 (s, 1H), 7.88 (d, 1H), 8.20 (d, 1H), 8.72 (s, 1H).

Retention Time (LC, method: ammonium acetate standard): 1.34 min.

MS (M+H⁺): 305.

Example 11N-[6-chloro-7-(2-dimethylamino-ethoxy)-9H-β-carbolin-8-yl]-2-methyl-nicotinamide

The desired compound was prepared according to Method A from6-chloro-7-(N,N)-dimethylaminoethoxy-9H-β-carboline-8-ylamine and2-methylnicotinic acid in a 40-60% yield.

¹H-NMR (300 MHz, DMSO-d₆): δ 1.92 (s, 6H), 2.49 (m, 2H), 2.68 (s, 3H),4.25 (m, 2H), 7.50 (dd, 1H), 8.16 (m, 2H), 8.38 (d, 1H), 8.42 (s, 1H),8.66 (m, 1H), 9.00 (s, 1H), 11.27 (s, 1H), 11.78 (s, 1H).

Retention Time (LC, method: ammonium acetate standard): 1.52 min.

MS (M+H⁺): 424.

Example 12 2-amino-cyclopentanecarboxylic acid(6-chloro-7-methoxy-9H-β-carbolin-8-yl)-amide

6-chloro-7-methoxy-9H-β-carboline-8-ylamine and2-tert-butoxycarbonylamino-cyclopentanecarboxylic acid were reactedusing Method A. To this product was added ˜5 ml of 4N HCl/dioxane andthe resulting mixture was allowed to stir at RT. The reaction wasfollowed by LC-MS until completion. Evaporation was allowed to removeall the solvent which gave a crude HCl salt. The desired product wasthen purified by preparative HPLC.

¹H-NMR (300 MHz, DMSO-d₆): δ 1.68 (m, 1H), 1.84 (m, 2H), 2.06 (m, 2H),2.20 (m, 1H), 3.46 (m, 1H), 3.67 (m, 1H), 3.89 (s, 3H), 8.24 (d, 2H),8.61 (d, 1H), 8.72 (s, 1H), 8.78 (d, 1H), 9.19 (s, 1H), 10.58 (s, 1H),13.20 (s, 1H).

Retention Time (LC, method: ammonium acetate standard): 1.54 min.

MS (M+H⁺): 359.

INTERMEDIATE 25:1-(2-dimethylamino-ethyl)-5-oxo-pyrrolidine-3-carboxylic acid

A mixture of commercially available itaconic acid andN,N-dimethylethylenediamine was heated up to 160° C. for about 20-25minutes. The mixture was allowed to cool to 100° C. and then dilutedwith MeOH to prevent solidification. The product was obtained in a 56%yield after crystallization from MeOH/EtOAc.

¹H-NMR (300 MHz, D₂O): δ 2.63 (dd, 1H), 2.80 (dd, 1H), 2.95 (s, 6H),3.15-3.25 (m, 1H), 3.32-3.44 (m, 1H), 3.44-3.76 (m, 4H), 3.82-3.94 (m,1H).

Retention Time (LC, method: ammonium acetate standard): 0.13 min.

MS (M+H⁺): 201.0.

Example 13 1-(2-Dimethylamino-ethyl)-5-oxo-pyrrolidine-3-carboxylic acid(6-chloro-9H-β-carbolin-8-yl)-amide

Prepared according to Method B from 6-chloro-9H-β-carboline-8-ylamineand 1-(2-dimethylamino-ethyl)-5-oxo-pyrrolidine-3-carboxylic acid in70-80% yield as the di-HCl salt.

¹H-NMR (300 MHz, D₂O): δ 2.97 (dd, 1H), 3.03 (s, 6H), 3.08 (dd, 1H),3.51 (t, 2H), 3.76-3.89 (m, 3H), 3.95 (dd, 1H), 4.01 (dd, 1H), 7.75 (d,1H), 8.24 (d, 1H), 8.45 (dd, 1H), 8.52 (dd, 1H), 9.10 (bs, 1H).

Retention Time (LC, method: ammonium acetate standard): 0.99 min.

MS (M+H⁺): 400.

Example 14 1-(2-dimethylamino-ethyl)-5-oxo-pyrrolidine-3-carboxylic acid(6-chloro-7-methoxy-9H-β-carbolin-8-yl)-amide

Prepared according to Method A from6-chloro-7-methoxy-9H-β-carboline-8-ylamine and1-(2-dimethylamino-ethyl)-5-oxo-pyrrolidine-3-carboxylic acid in 60%yield following purification using a semi-preparative Chiralcel ODcolumn with 85/7.5/7.5 hexane/EtOH/MeOH as the eluant.

¹H-NMR (300 MHz, DMSO-d₆): δ 2.44 (s, 6H), 2.75 (t, 2H), 2.88 (d, 2H),3.56 (t, 2H), 3.66 (m, 1H), 3.85 (m, 2H), 3.90 (s, 3H), 8.03 (d, 1H),8.21 (s, 1H), 8.28 (d, 1H), 8.79 (s, 1H).

Retention Time (LC, method: ammonium acetate standard): 1.42 min.

MS (M+H⁺): 430.

INTERMEDIATE 26: 6-chloro-7-fluoro-9H-β-carbolin-8-ylamine

A slurry of 6-chloro-7-fluoro-8-nitro-9H-β-carboline (500 mg, 1.88 mmol)in MeOH (25 ml) was degassed with argon. Palladium on charcoal (20% w/won C, 50 mg) was added and the reaction vessel was flushed withhydrogen. The slurry was stirred under a balloon of hydrogen for 6 hr,then filtered through celite and concentrated under reduced pressure toyield 6-chloro-7-fluoro-9H-β-carbolin-8-ylamine (400 mg) as a brownsolid.

¹H-NMR (300 MHz, DMSO-d₆): δ 11.48 (br s, 1); 8.99-8.98 (m, 1);8.37-8.35 (m, 1); 8.11-8.09 (m, 1); 7.74-7.72 (m, 1); 5.65 (br s, 2).

HCOOH standard conditions.

DAD R_(f)=1.00 min.

MS (M+H⁺): 236.

Example 15N-(6-chloro-7-fluoro-9H-β-carbolin-8-yl)-2-methyl-nicotinamide

A solution of 6-chloro-7-fluoro-9H-β-carbolin-8-ylamine (100 mg, 0.424mmol) in pyridine (2.5 ml) was stirred at RT. 2-methyl nicotinic acid(70 mg, 0.509 mmol) was added, followed by EDCI (130 mg, 0.678 mmol).The suspension was stirred at 100° C. for a day. The pyridine wasremoved under reduced pressure and the resulting dark oil was trituratedwith saturated aqueous NaHCO₃. The precipitate which formed was filteredand washed with MeOH. The material was treated with 2M HCl in Et₂O toyield a gray solid, the di-HCl saltN-(6-chloro-7-fluoro-9H-β-carbolin-8-yl)-2-methyl-nicotinamide (110 mg).

¹H-NMR (300 MHz, DMSO-d₆): δ 13.15 (s, 1); 11.03 (s, 1); 9.33 (s, 1);8.99-8.97 (m, 1); 8.92-8.89 (m, 1); 8.79-8.73 (m, 2); 8.50 (m, 1); 7.70(m, 1); 2.80 (s, 3).

HCOOH standard conditions.

DAD R_(f)=0.91 min.

MS (M+H⁺): 355.

INTERMEDIATE 27: 6-chloro-7-methylsulfanyl-8-nitro-9H-β-carboline

A 250 ml round-bottom flask with magnetic stirrer was charged with6-chloro-7-fluoro-8-nitro-β-carboline (Intermediate 5, 3.959 g, 14.9mmol) and 100 ml anhydrous DMF. The resulting orange mixture was cooledto 0° C. (ice and water bath) and sodium thiomethoxide (1.809 g, 25.8mmol) in powder form was added slowly thereto. The reaction mixture wasstirred for 1 hr at 0° C., warmed to RT, and added slowly to a stirringmixture of 4:1 H₂O/saturated aqueous sodium bicarbonate (500 ml). Theprecipitated solid was collected via suction filtration and air-dried toafford 4.017 g of 6-chloro-7-methylsulfanyl-8-nitro-9H-β-carboline as anorange powder. The crude material was used directly in subsequent steps.

¹H-NMR (300 MHz, Methanol-d₄): δ 8.91 (1H, d) 8.63 (1H, s) 8.42 (1H, d)8.17 (1H, dd) 2.54 (3H, s).

LCMS (formic acid standard method) retention time=1.43 min.

MS (M+H⁺): 294.

INTERMEDIATE 28: 6-chloro-7-methylsulfanyl-9H-β-carbolin-8-ylamine

A 500 ml round-bottom flask with magnetic stirrer was charged with6-chloro-7-methylsulfanyl-8-nitro-9H-β-carboline (4.011 g, 13.6 mmol)and 200 ml anhydrous ethanol. To the resulting mixture was added aqueousammonium chloride (75 ml of 0.33 M solution, 24.7 mmol), aqueoushydrochloric acid (10 ml of 1 M solution, 10 mmol), and iron powder(7.734 g, 138 mmol). The resulting mixture was heated to 60° C. (oilbath) and stirred vigorously for 3.5 hr. The reaction was cooled to RT,diluted with EtOAc (75 ml) and activated charcoal (ca. 2.5 g) was added.The resulting mixture was stirred at RT for an additional 1.5 hr,filtered through a pad of celite, and the resulting filtrateconcentrated (rotavap, then vacuum pump) to afford 5.153 g of ayellowish-orange colored solid. The solid was redissolved in MeOH(50-100 ml) and slowly added to saturated aqueous sodium bicarbonate(500 ml) with stirring. The mixture was stirred at RT for 45 min and theresulting solid collected via suction filtration and air-dried to afford3.476 g of 6-chloro-7-methylsulfanyl-9H-β-carbolin-8-ylamine as a tansolid which was used without further purification in subsequent steps.

¹H-NMR (300 MHz, Methanol-d₄): δ 8.87 (1H, s) 8.35-8.24 (1H, m)8.16-8.06 (1H, m) 7.67 (1H, s) 2.32 (3H, s).

LCMS (ammonium acetate standard method) retention time=2.12 min.

MS (M+H⁺): 264.

Example 16N-(6-chloro-7-methylsulfanyl-9H-β-carbolin-8-yl)-2-methyl-nicotinamide

A 250 ml round-bottom flask with magnetic stirrer was charged with6-chloro-7-methylsulfanyl-9H-β-carbolin-8-ylamine (Intermediate 28,2.336 g, 8.86 mmol) and 2-methylnicotinic acid (3.219 g, 23.4 mmol) in80 ml anhydrous pyridine. To the resulting reaction mixture at RT wasadded solid 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(7.080 g, 36.9 mmol) and the reaction mixture was heated to 100° C. (oilbath) for 2 days. The resulting mixture was cooled to RT andconcentrated (rotavap) to afford a brown residue. The residue wasredissolved in MeOH (50 ml), slowly added to a stirring mixture of 5:1H₂O/saturated aqueous sodium bicarbonate (600 ran) and stirred at RT for˜18 hr. The precipitated solid was collected via suction filtration,washed with Et₂O (2×150 ml), and air-dried to afford 3.036 g of crudeN-(6-chloro-7-methylsulfanyl-9H-β-carbolin-8-yl)-2-methyl-nicotinamideas a brown solid. The crude material was purified via HPLC(yields=˜40-60%)

¹H-NMR (300 MHz, DMSO-d₆): δ 11.72 (1H, s) 10.50 (1H, s) 8.97 (1H, s)8.62 (1H, dd) 8.57 (1H, s) 8.41 (1H, d) 8.33-8.27 (1H, m) 8.21 (1H, d)2.73 (3H, s) 2.41 (3H, s).

LCMS (ammonium acetate standard method) retention time=1.89 min.

MS (M+H⁺): 383.

INTERMEDIATE 29: 6-chloro-7-ethylsulfanyl-8-nitro-9H-β-carboline

A 25 ml round-bottom flask with a magnetic stirrer was charged with6-chloro-7-fluoro-8-nitro-9H-βcarboline (102 mg, 0.38 mmol) in 5 ml ofanhydrous DMF. To the resulting orange mixture at RT was slowly addedsodium thioethoxide (80% pure, 69.7 mg, 0.66 mmol) in powder form. Thereaction mixture was stirred at RT for 45 minutes, then added drop-wiseto a 5:1 mixture of H₂O/saturated sodium bicarbonate (˜30 ml). Theresulting precipitated solid was collected by suction filtration, washedwith 1:1 hexanes/diethyl ether (2×20 ml), and air-dried to afford 95.0mg of 6-chloro-7-ethylsulfanyl-8-nitro-9H-β-carboline as an orange solid(79%).

¹H-NMR (300 MHz, CD₃OD, ppm) δ 8.91 (1H, s) 8.63 (1H, s) 8.42 (1H, d)8.18 (1H, d) 3.04 (2H, q) 1.20 (3H, t).

Retention Time (LC, formic acid standard method): 1.71 min.

MS (M+H⁺): 308.

INTERMEDIATE 30: 6-chloro-7-ethylsulfanyl-9H-β-carbolin-8-ylamine

A 50 ml round-bottom flask with magnetic stirrer was charged with6-chloro-7-ethylsulfanyl-8-nitro-9H-β-carboline (85.0 mg, 0.28 mmol) in10 ml anhydrous ethanol. To the resulting orange mixture at RT was added0.33 M aqueous ammonium chloride (2.0 ml, 0.66 mmol) and iron powder(680 mg, 12.2 mmol). The reaction mixture was heated to 60° C. andstirred vigorously for 20 hr. Next, the mixture was cooled to RT,diluted with ethyl acetate (15 ml), and activated charcoal (˜180 mg) wasadded. The resulting mixture was filtered through a pad of celite andthe filtrate was concentrated to afford 77.8 mg of6-chloro-7-ethylsulfanyl-9H-β-carbolin-8-ylamine as a yellow solid(>99%).

¹H-NMR (300 MHz, CD₃OD, ppm) δ 8.94 (1H, s) 8.33-8.29 (1H, m) 8.21-8.18(1H, m) 7.73 (1H, s) 2.85 (2H, q) 1.21 (3H, t).

LCMS (ammonium acetate standard method) retention time 2.13 min.

(M⁺=278; M⁻=276).

Example 17N-(6-chloro-7-ethylsulfanyl-9H-β-carbolin-8-yl)-2-methyl-nicotinamide

A 25 ml round-bottom flask with magnetic stirrer was charged with6-chloro-7-ethylsulfanyl-9H-β-carbolin-8-ylamine (37.2 mg, 0.13 mmol)and 2-methylnicotinic acid (36.2 mg, 0.26 mmol) in 3 ml anhydrouspyridine. To the resulting light-orange mixture at RT was added solid1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (73.2 mg,0.38 mmol) and the resulting reaction mixture was heated to 80° C. for 5days. Next, the reaction mixture was cooled to RT and concentrated toafford a brown, viscous syrup. The syrup was dissolved in a minimalamount of MeOH (˜2 ml), slowly added to a 5:1 mixture of H₂O/saturatedsodium bicarbonate (˜20 ml), and stirred at RT for 2.5 hr. The resultingprecipitated solid was collected via suction filtration, washed with 1:1hexanes/diethyl ether (2×20 ml), and air-dried to afford 21.0 mg ofN-(6-chloro-7-ethylsulfanyl-9H-β-carbolin-8-yl)-2-methyl-nicotinamide asa tan solid (−38%). LCMS (ammonium acetate standard method) retentiontime=2.33 min. (M⁺=397; M⁻=395).

¹H-NMR (300 MHz, CD₃OD, ppm) δ 8.89 (1H, s) 8.63-8.58 (1H, m) 8.44-8.38(2H, m) 8.36 (1H, d) 8.15 (1H, d) 7.52-7.44 (1H, m) 2.98 (2H, q) 2.84(3H, s) 1.20 (3H, t).

INTERMEDIATE 31:[2-(6-chloro-8-nitro-9H-β-carbolin-7-ylsulfanyl)-ethyl]-dimethyl-amine

A 25 ml round-bottom flask with magnetic stirrer was charged with6-chloro-7-fluoro-8-nitro-9H-β-carboline (98 mg, 0.37 mmol) in 2 ml ofanhydrous DMF. A second 10 ml round-bottom flask with magnetic stirrerwas charged with 2-dimethylamino-ethanethiol hydrochloride (100 mg, 0.70mmol) in 2 ml anhydrous DMF. To the resulting suspension was addedn-butyllithium (0.43 ml of 1.6 M solution in hexanes, 0.69 mmol) viasyringe and the mixture was stirred for 5 min at RT. Next, the thioanionsolution was added via syringe to6-chloro-7-fluoro-8-nitro-9H-β-carboline, and the resulting red solutionwas stirred at RT for 30 min. The reaction mixture was slowly added to a5:1 mixture of H₂O/saturated aqueous sodium bicarbonate (30 ml) andallowed to sit at RT for several hours. The resulting precipitated solidwas collected via suction filtration and air-dried to afford 109 mg of[2-(6-chloro-8-nitro-9H-β-carbolin-7-ylsulfanyl)-ethyl]-dimethyl-amineas an orange solid (83%).

LCMS (ammonium acetate standard method) retention time=1.35 min.

(M⁺=351; M⁻=349).

¹H-NMR (300 MHz, CD₃OD, ppm) δ 8.92 (1H, d, J=1.0 Hz) 8.66 (1H, s) 8.43(1H, d) 8.19 (1H, dd) 3.18-3.13 (2H, m) 2.57-2.52 (2H, m) 2.21 (6H, s).

INTERMEDIATE 32:6-chloro-7-(2-dimethylamino-ethylsulfanyl)-9H-β-carbolin-8-ylamine

A 50 ml round-bottom flask with a magnetic stirrer was charged with[2-(6-chloro-8-nitro-9H-β-carbolin-7-ylsulfanyl)-ethyl]-dimethyl-amine(106 mg, 0.30 mmol) in 8 ml of anhydrous ethanol. To the resultingorange mixture at RT was added 0.33 M aqueous ammonium chloride (1.95ml, 0.64 mmol) and iron powder (540 mg, 9.67 mmol). The reaction mixturewas heated to 60° C. and stirred vigorously for 20 hr. Next, the mixturewas cooled to RT, diluted with ethyl acetate (20 ml) and activatedcharcoal (ca. 150 mg) was added. The resulting mixture was filteredthrough a pad of celite and the resulting filtrate concentrated toafford 103 mg of6-chloro-7-(2-dimethylamino-ethylsulfanyl)-9H-β-carbolin-8-ylamine as ayellow solid. The crude product was used directly in the coupling step.

LCMS (ammonium acetate standard method) retention time=1.34 min.

(M⁺=321; M⁻=319).

¹H-NMR (300 MHz, CD₃OD, ppm) δ 8.87 (1H, s) 8.30 (1H, d) 8.06 (1H, d)7.75 (1H, s) 3.23-3.13 (4H, m) 2.84 (6H, s).

Example 18N-[6-chloro-7-(2-dimethylamino-ethylsulfanyl)-9H-β-carbolin-8-yl]-2-methyl-nicotinamide

A 25 ml round-bottom flask with magnetic stirrer was charged with6-chloro-7-(2-dimethylamino-ethylsulfanyl)-9H-β-carbolin-8-ylamine (45.2mg, 0.14 mmol) and 2-methylnicotinic acid (39.0 mg, 0.28 mmol) in 4.5 mlof anhydrous pyridine. To the resulting light-orange mixture at RT wasadded solid 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(95.0 mg, 0.49 mmol). The resulting reaction mixture was heated to 80°C. for 3 days. Next, the reaction was cooled to RT and concentrated toafford a brown, viscous syrup. The syrup was dissolved in a minimalamount of MeOH (˜2 ml), slowly added to a 5:1 mixture of H₂O/saturatedsodium bicarbonate (˜25 ml), and extracted with ethyl acetate (3×30 ml).The combined organic layers were washed with brine (1×30 ml), dried overNa₂SO₄, filtered and concentrated to afford a brown residue (79.6 mg).The residue was redissolved in MeOH (˜5 ml), and filtered through acotton plug. To the resulting filtrate was added HCl in 1,4-dioxane (1.0ml, 4.0 mmol), the resulting solution stirred at RT for 3 hr, and addeddrop-wise to diethyl ether (30 ml) with stirring. The resultingprecipitated product was collected via suction filtration, washed withether and air-dried to afford 36.3 mg ofN-[6-chloro-7-(2-dimethylamino-ethylsulfanyl)-9H-β-carbolin-8-yl]-2-methyl-nicotinamidetris-hydrochloride as a yellow powder.

¹H-NMR (300 MHz, CD₃OD, ppm) δ 8.95 (1H, s) 8.62 (1H, dd) 8.43 (1H, s)8.38-8.34 (2H, m) 8.16 (1H, d) 7.50 (1H, dd) 3.09 (2H, t) 2.85 (3H, s)2.30 (2H, t) 1.99 (6H, s).

LCMS (ammonium acetate standard method) retention time=1.56 min.

(M⁺=440; M⁻=438).

INTERMEDIATE 33: morpholine-3(S),4-dicarboxylic acid 4-tert-butyl ester

A suspension of morpholine-3(S)-carboxylic acid (2.00 g, 15.3 mmol) inDMF (75 ml) was stirred at RT. Triethylamine (7.47 ml, 53.6 mmol) anddi-tert-butyl dicarbonate (BOC₂O, 4.02 g, 18.4 mmol) were added. Thesuspension was stirred at RT for one hour, during which time thereaction formed a clear yellow solution. The solution was concentratedto a reduced volume (˜25 ml) and diluted with water (15 ml) and 1N HCl(15 ml). The mixture was poured into a separatory funnel, dilutedfurther with water (100 ml) and brine (100 ml), and extracted with Et₂O(3×100 ml). The organic layer was washed with brine, dried, filtered andconcentrated to yield a white solid.

The solid, which contained excess BOC₂O, was dissolved in Et₂O (500 ml)and extracted with 1N NaOH (3×100 ml). The aqueous layer was acidifiedwith 6N HCl to approximately a pH of 2, then extracted quickly with Et₂O(3×100 ml). The Et₂O layer was dried, filtered and concentrated to yieldwhite solid (3.07 g).

¹H-NMR (300 MHz, DMSO-d₆): δ 12.95 (br s, 1); 4.34-4.30 (m, 1);4.18-4.10 (m, 1); 3.83-3.74 (m, 1); 3.59-3.51 (m, 2); 3.39-3.32 (m, 1);3.21-2.95 (m, 1); 1.41-1.36 (m, 9).

NH₄OAc standard conditions.

ELSD R_(f)=1.08 min.

M−H=230.

Example 19 4-methyl-morpholine-3(S)-carboxylic acid(6-chloro-9H-β-carbolin-8-yl)-amide

A slurry of morpholine-3(S)-carboxylic acid (3.00 g, 22.9 mmol) in EtOH(115 ml) was stirred at RT. A solution of aqueous CH₂O (3.42 ml, 45.8mmol, 37% w/w in H₂O) was added, followed by Pd(OH)₂ (600 mg, 20% w/w oncharcoal). The flask was charged with hydrogen (1 atm) and the greyslurry was stirred for 24 hr at RT under a balloon of hydrogen. Theflask was purged with nitrogen and the black slurry was diluted withMeOH, filtered through filter paper and concentrated to a reducedvolume. The pale grey solution was filtered through a 0.45 μm syringefilter to remove residual Pd(OH)₂ and concentrated to yield a clearcolorless oil. The oil was placed under high vacuum for 24 hr and awhite, solid foam was isolated. The foam was dissolved in pyridine (200ml) and 6-chloro-9H-β-carbolin-8-ylamine (3.74 g, 17.2 mmol) was added,followed by EDCI (5.87 g, 30.6 mmol). The clear pale orange solution wasstirred at RT for 24 hr. The solution was diluted with H₂O (300 ml) andpoured into a separatory funnel containing EtOAc (300 ml). The mixturewas shaken and the layers were separated. The aqueous layer wasextracted with EtOAc (3×150 ml) and the combined organic layers werewashed with H₂O and brine. The organic layer was dried, filtered andconcentrated to yield a brown oil which was placed under high vacuum.The resulting brown foam was triturated with MeOH and the precipitatewhich formed was filtered and washed with MeOH. The resulting paleyellow solid was purified via chiral HPLC to yield a white solid (3.23g).

¹H-NMR (300 MHz, DMSO-d₆): δ 11.36 (s, 1); 10.02 (s, 1); 9.04 (s, 1);8.38 (d, 1); 8.22-8.21 (m, 1); 8.15 (d, 1); 7.91-7.90 (m, 1); 4.00 (dd,1); 3.85-3.81 (m, 1); 3.69-3.58 (m, 2); 2.99-2.95 (dd, 1); 2.89-2.85 (m,1); 2.32 (s, 3); 2.32-2.24 (m, 1). NH₄OAc standard conditions.

DAD R_(f)=1.89 min.

MFH=345.

Chiral preparative HPLC: 10% v/v EtOH/Hexanes.

Chiralcel OD column.

R_(f)=11.5-14 min.

Enantiopurity of product≧99% ee.

METHOD C: PROCEDURE FOR 4-MORPHOLINE SUBSTITUTED ANALOGS

AS outlined for Intermediate 34, Intermediate 35 and Example 20:

INTERMEDIATE 34:3(S)-(6-chloro-9H-β-carbolin-8-ylcarbamoyl)-morpholine-4-carboxylic acidtert-butyl ester

A solution of morpholine-3(S),4-dicarboxylic acid 4-tert-butyl ester(2.83 g, 12.7 mmol) in pyridine (106 ml) was stirred at RT.6-chloro-9H-β-carbolin-8-ylamine (2.30 g, 10.6 mmol) was added, followedby EDCI (4.06 g, 21.2 mmol). The clear orange-to-brown solution wasstirred at RT for 14 hr. The solution was diluted with H₂O (120 ml) andpoured into a separatory funnel containing EtOAc (200 ml), H₂O (100 ml)and brine (100 ml). The mixture was shaken and the layers wereseparated. The aqueous layer was extracted with EtOAc (2×50 ml) and thecombined organic layers were washed with brine. The organic layer wasdried, filtered and concentrated to a reduced volume, then addeddrop-wise to a stirring solution of 1:1 Et₂O/hexanes (500 ml). Theprecipitate which formed was filtered and washed with 1:1 Et₂O/Hexanes.The filtrate was concentrated to a reduced volume and a second crop ofprecipitate was collected. The solid product was placed under highvacuum for 2 hr to yield3(S)-(6-chloro-9H-(3-carbolin-8-ylcarbamoyl)-morpholine-4-carboxylicacid tert-butyl ester as a pale yellow to pale brown solid (4.36 g).

¹H-NMR (300 MHz, DMSO-d₆): δ 11.30 (s, 1); 10.13 (s, 1); 9.06 (s, 1);8.40-8.38 (m, 1); 8.19-8.16 (m, 2); 7.98 (s, 1); 4.67-4.47 (m, 2);3.96-3.60 (m, 2); 3.64-3.39 (m, 3); 1.42 (s, 9).

NH₄OAc standard conditions.

DAD R_(f)=2.31 min.

M+H=431.

INTERMEDIATE 35:2(R)-[3(S)-(6-chloro-9H-β-carbolin-8-ylcarbamoyl)-morpholin-4-ylmethyl]-pyrrolidine-1-carboxylicacid tert-butyl ester

A solution of3(S)-(6-chloro-9H-β-carbolin-8-ylcarbamoyl)-morpholine-4-carboxylic acidtert-butyl ester (1.00 g, 2.32 mmol) in CH₂Cl₂ (6 ml) was stirred at RT.Trifluoroacetic acid (6 ml) was added and the solution was stirred at RTfor 45 min, then concentrated to a residue. The residue was concentratedonce more from CH₂Cl₂ to yield a yellow-brown solid which was dissolvedin THF (13 ml) under argon. Gentle warming was sometimes needed toensure complete dissolution. A solution ofN-(tert-butoxycarbonyl)-D-prolinal (693 mg, 3.48 mmol) in THF (2 ml) wasadded, followed by sodium triacetoxyborohydride (738 mg, 3.48 mmol). Thesolution was stirred at RT for 30 min, then quenched via addition of 1Naqueous NaOH (30 ml). The mixture was poured into a separatory funnelcontaining EtOAc (100 ml), H₂O (100 ml), and brine (100 ml). The mixturewas shaken and the layers were separated. The aqueous layer wasextracted with EtOAc (2×50 ml) and the combined organic layers werewashed with brine. The organic layer was dried, filtered andconcentrated to yield a light brown solid. Column chromatography (2%-4%MeOH/CH₂Cl₂) yielded2(R)-[3(S)-(6-Chloro-9H-β-carbolin-8-ylcarbamoyl)-morpholin-4-ylmethyl]-pyrrolidine-1-carboxylicacid tert-butyl ester as a white solid (915 mg).

¹H-NMR (300 MHz, DMSO-d₆): δ 11.30 (s, 1); 9.88 (s, 1); 9.04 (s, 1);8.39-8.37 (m, 1); 8.20-8.15 (m, 2); 7.95 (s, 1); 3.99-3.82 (m, 3);3.69-3.63 (m, 2); 3.44-3.32 (m, 1); 3.27-3.11 (m, 3); 2.92-2.80 (m, 1);2.44-2.32 (m, 1); 1.99-1.67 (m, 5); 1.33 (s, 9).

HCOOH standard conditions.

DAD R_(f)=1.39 min.

M+H=514.

Chiral HPLC.

The enantiopurity of the sample was checked. The samples were ≧97% ee.

Chiralpak AD column.

15% v/v EtOH/Hexanes containing 0.1% Et₂NH.

Example 20 4-pyrrolidin-2(R)-ylmethyl-morpholine-3(S)-carboxylic acid(6-chloro-9H-β-carbolin-8-yl)-amide, HCl salt)

To a solution of2(R)-[3(S)-(6-chloro-9H-β-carbolin-8-ylcarbamoyl)-morpholine-4-ylmethyl]-pyrrolidine-1-carboxylicacid tert-butyl ester (850 mg, 1.65 mmol) in MeOH (16 ml) was addedconcentrated aqueous HCl (13 ml). The solution was stirred at RT for30-45 min, during which time a fine yellow precipitate formed. Thereaction mixture was concentrated to yield4-pyrrolidin-2(R)-ylmethyl-morpholine-3(S)-carboxylic acid(6-chloro-9H-β-carbolin-8-yl)-amide, HCl salt) as a pale yellow solid(755 mg).

¹H-NMR (300 MHz, DMSO-d₆): δ 13.30 (s, 1); 11.56 (br s, 1); 9.63 (br s,1); 9.46 (s, 1); 8.87-8.85 (m, 1); 8.66-8.57 (m, 2); 8.25 (s, 1);4.38-4.26 (m, 1); 4.24-4.08 (m, 1); 4.04-3.86 (m, 2); 3.86-3.68 (m, 2);3.57-3.39 (m, 1); 3.39-3.02 (m, 4); 2.99-2.76 (m, 1); 2.10-1.84 (m, 3);1.75-1.56 (m, 1).

HCOOH standard conditions.

DAD R_(f)=0.81 min.

M+H=414.

INTERMEDIATE 36: cis-2-(tert-butoxycarbonylamino)-cyclopentanecarboxylicacid (6-chloro-9H-carbolin-8-yl) amide

A solution of cis-2-(tert-butoxycarbonylamino)-cyclopentane carboxylicacid (550 mg, 2.4 mmol) in pyridine (10 ml) was stirred at RT.6-chloro-9H-β-carbolin-8-ylamine (436 mg, 2.0 mmol) was added, followedby EDCI (615 mg, 3.2 mmol), and the orange solution was stirred at RTfor 1.5 hr. The solution was diluted with H₂O (20 ml) and poured into aseparatory funnel containing H₂O (50 ml) and EtOAc (100 ml). The mixturewas shaken and the layers were separated. The aqueous layer wasextracted with EtOAc (100 ml). The combined organic layers were washedwith brine, dried over MgSO₄, filtered, and concentrated to orange oilysolids which were subsequently triturated with 5% MeOH in Et₂O (20 ml)and captured by filtration to yieldcis-2-(tert-butoxycarbonylamino)-cyclopentanecarboxylic acid(6-chloro-9H-carbolin-8-yl) amide as a light yellow solid (740 mg).

¹H-NMR (300 MHz, DMSO-d₆): δ 11.17 (s, 1); 9.89 (s, 1); 9.04 (s, 1);8.36 (d, 1); 8.18-8.08 (m, 2); 7.95 (s, 1); 6.92 (d, 1); 4.32-4.22 (m,1); 3.16-3.09 (m, 1); 2.13-2.01 (m, 1); 1.96-1.75 (m, 3); 1.74-1.59 (m,1); 1.58-1.42 (m, 1); 1.07 (s, 9).

NH₄OAc standard conditions.

DAD R_(f)=2.52 min.

M+H=429.

Example 21 cis-2-amino-cyclopentanecarboxylic acid(6-chloro-9H-β-carbolin-8-yl)-amide

A solution of 2-(tert-butoxycarbonylamino)-cyclopentanecarboxylic acid(6-chloro-9H-β-carbolin-8-yl) amide (736 mg, 1.72 mmol) intrifluoroacetic acid (5 ml) was stirred at RT for 20 min, thenconcentrated to an orange oil. The oil was dissolved in MeOH (5 ml) andneutralized with a saturated aqueous sodium bicarbonate solution (25ml). The resulting mixture was further diluted with H₂O (25 ml) andEtOAc (100 ml). The aqueous layer was removed and extracted with EtOAc(100 ml). The organic layers were combined, washed with brine, driedover MgSO₄, filtered and concentrated to yellow solids (507 mg), Thesesolids were dissolved in MeOH (5 ml) and a solution of HCl in dioxane (4M, 1.5 ml) was added. The bright yellow solution was stirred 30 min,then concentrated to yield cis-2-amino-cyclopentanecarboxylic acid(6-chloro-9H-β-carbolin-8-yl)-amide as a yellow powder (600 mg).

¹H-NMR (300 MHz, DMSO-d₆): δ 9.29 (s, 1); 8.75 (d, 1); 8.53 (d, 1); 8.37(s, 1); 8.02 (s, 1); 4.05-3.95 (m, 1); 3.42-3.34 (m, 1); 2.46-1.80 (m,6).

NH₄OAc standard conditions.

DAD R_(f)=1.65 min.

M+H=329.

Example 22 4-(2-amino-ethyl)-morpholine-3-carboxylic acid(6-chloro-9H-β-carbolin-8-yl)-amide, HCl salt

Method C was followed using racemic morpholine-3-carboxylic acidreductively alkylated with 2-aminoacetaldehyde.

¹H-NMR (300 MHz, MeOH-d₄): δ 9.37 (s, 1); 8.76 (d, 1); 8.55 (d, 1); 8.44(d, 1); 8.06 (d, 1); 4.68-4.55 (m, 2); 4.17-3.99 (m, 3); 3.84-3.73 (m,2); 3.57-3.39 (m, 4).

NH₄OAc standard conditions.

DAD R_(f)=1.69 min.

M+H=374.

Example 23 4-(2(S)-amino-propyl)-morpholine-3(S)-carboxylic acid(6-chloro-9H-β-carbolin-8-yl)-amide, HCl salt (first elutingdiastereomer)

Method C was followed using racemic morpholine-3-carboxylic acidreductively alkylated with the appropriate alanine aldehyde. Thediastereomers were separated via column chromatography prior to thedeprotection step.

¹H-NMR (300 MHz, DMSO-d₆): δ 9.32 (s, 1); 8.76 (d); 8.55 (d, 1); 8.41(s, 1); 8.08 (s, 1); 4.38 (d, 1); 4.32-4.21 (m, 1); 4.16-4.09 (m, 1);4.04-3.95 (m, 2); 3.79-3.57 (m, 2); 3.47-3.40 (m, 1); 3.22-3.05 (m, 2);1.44 (d, 3).

NH₄OAc standard conditions.

DAD R_(f)=1.38 min.

M+H=388.

Example 24 4-(2(S)-amino-propyl)-morpholine-3(R)-carboxylic acid(6-chloro-9H-β-carbolin-8-yl)-amide, HCl salt (second elutingdiastereomer)

Method C was followed using racemic morpholine-3-carboxylic acidreductively alkylated with the appropriate alanine aldehyde. Thediastereomers were separated via column chromatography prior to thedeprotection step.

¹H-NMR (300 MHz, DMSO-d₆): δ 9.34 (s, 1); 8.77 (d, 1); 8.55 (d, 1); 8.42(s, 1); 8.06 (s, 1); 4.42 (d, 1); 4.30-4.12 (m, 1); 4.07-3.92 (m, 3);3.89-3.74 (m, 1); 3.65-3.49 (m, 1); 3.25-2.90 (m, 3); 1.36 (d, 3).

NH₄OAc standard conditions.

DAD R_(f)=1.57 min.

M+H=388.

Example 25 2-amino-cyclohexanecarboxylic acid(6-chloro-9H-β-carbolin-8-yl)-amide

A solution of cis-2-(tert-butoxycarbonylamino)-cyclohexane carboxylicacid (255 mg, 1.05 mmol) in pyridine (10 ml) was stirred at RT.6-chloro-9H-β-carbolin-8-ylamine (218 mg, 1.00 mmol) was added, followedby EDCI (315 mg, 1.64 mmol) and the slightly turbid pale orange solutionwas stirred at RT for 16 hr. The solution was diluted with H₂O (20 ml)and poured into a separatory funnel containing H₂O (50 ml) and EtOAc (50ml). The mixture was shaken and the layers were separated. The aqueouslayer was extracted with EtOAc (50 ml) and the combined organic layerswere washed with brine. The organic layer was dried, filtered andconcentrated to yield a yellow oil which was placed under high vacuumfor 4 hr. The resulting yellow-brown glass was slurried in CH₂Cl₂ (10ml) at RT. Trifluoroacetic acid (5 ml) was added and the slurryinstantly dissolved to form a clear orange solution. The solution wasstirred at RT for 45 min, then concentrated to a brown residue. Theresidue was azeotroped from toluene (3×10 ml) to yield a yellow solid. Asolution of dilute aqueous Na₂CO₃ was prepared by adding a small volumeof 10% aqueous Na₂CO₃ to H₂O (50 ml) until the aqueous solution reacheda pH of 10. The yellow solid was dissolved in minimal MeOH and was addeddrop-wise to the aqueous solution with stirring. The precipitate whichformed was filtered, washed with H₂O and placed under high vacuum toyield 2-amino-cyclohexanecarboxylic acid(6-chloro-9H-β-carbolin-8-yl)-amide as pale yellow solid (147 mg).

¹H-NMR (300 MHz, DMSO-d₆): δ 9.00 (s, 1); 8.37-8.34 (m, 1); 8.16-8.13(m, 2); 7.83 (m, 1); 5.66-5.00 (br s, 2); 3.42-3.40 (m, 1); 2.70-2.62(m, 1); 2.02-1.90 (m, 1); 1.70-1.54 (m, 5); 1.42-1.29 (m, 2). NH₄OAcstandard conditions.

DAD R_(f)=1.46 min.

M+H=343.

Example 26 4-(2(R)-amino-propyl)-morpholine-3(S)-carboxylic acid(6-chloro-9H-β-carbolin-8-yl)-amide, HCl salt

Method C was followed using racemic morpholine-3-carboxylic acidreductively alkylated with the appropriate alanine aldehyde. Thediastereomers were separated via column chromatography prior to thedeprotection step.

¹H-NMR (300 MHz, DMSO-d₆): δ 9.35 (s, 1); 8.77 (m, 1); 8.55 (m, 1); 8.43(s, 1); 8.01 (s, 1); 4.45 (d, 1); 4.26 (m, 1); 4.09-3.91 (m, 3); 3.79(m, 1); 3.63 (m, 1); 3.28-2.99 (m, 3); 1.37 (d, 3).

NH₄OAc standard conditions.

DAD R_(f)=1.39 min.

M+H=388.

Example 27 4-(2(R)-amino-3-phenyl-propyl)-morpholine-3(S)-carboxylicacid (6-chloro-9H-β-carbolin-8-yl)-amide, HCl salt

Method C was followed using racemic morpholine-3-carboxylic acidreductively alkylated with the appropriate alanine aldehyde. Thediastereomers were separated via column chromatography prior to thedeprotection step.

¹H-NMR (300 MHz, DMSO-d₆): δ 9.34 (s, 1); 8.77 (d, 1); 8.55 (d, 1); 8.42(s, 1); 8.04 (s, 1); 7.44-7.23 (m, 5); 4.39 (d, 1); 4.65-4.07 (m, 1);4.40-3.82 (m, 4); 3.50-3.25 (m, 1); 3.30-3.14 (m, 1); 3.11-2.88 (m, 3);2.85-2.69 (m, 1).

NH₄OAc standard conditions.

DAD R_(f)=1.90 min.

M+H=464.

METHOD D: CHROMATOGRAPHY CONDITIONS LCMS

Column type: Phenomenex Luna C18(2) columns, 5 um, size 50×4.6 mmRun time: 5.00 minute run

NH₄OAc Conditions:

Solvent A: 10 mM NH₄OAc 99% H₂O 1% MeCN Solvent B: 10 mM NH₄OAc 5% H2O95% MeCN

Standard Gradient:

Initial conditions—95% A, 5% B3.5 minute gradient from 5%-100% B3.5-4.3 minutes hold at 100% B4.3-5 minutes initial conditions

Polar Gradient:

Initial conditions—70% A, 30% B3.5 minute gradient from 70%-100% B3.5-4.3 minutes hold at 100% B4.3-5 minutes initial conditions

Nonpolar Gradient:

Initial conditions—100% A3.5 minute gradient from 0%-50% B3.5-4.3 minutes hold at 100% B4.3-5 minutes initial conditions

HCOOH Conditions:

Solvent C, 0.1% HCOOH 99% H₂O 1% MeCN Solvent D: 0.1% HCOOH 5% H₂O 95%MeCN

Standard Gradient:

Initial conditions—95% C, 5% D3.5 minute gradient from 5%-100% D3.5-4.3 minutes hold at 100% D4.3-5 minutes initial conditions

Polar Gradient:

Initial conditions—70% C, 30% D3.5 minute gradient from 70%-100% D3.5-4.3 minutes hold at 100% D4.3-5 minutes initial conditions

Nonpolar Gradient:

Initial conditions—100% C3.5 minute gradient from 0%-50% D3.5-4.3 minutes hold at 100% D4.3-5 minutes initial conditions

INTERMEDIATE 37: 6-chloro-2,3,4,9-tetrahydro-1H-β-carboline, HCl salt

5-chlorotryptamine hydrochloride (5 g, 20 mmol, 1 equiv.) was dissolvedin 40 ml 3 M NaOAc buffer (pH=4.8) and 40 ml water. Glyoxalic acid (1.84g, 20 mmol, 1 equiv.) was added in one portion and the solution wasstirred at RT overnight. The resulting thick slurry was filtered and thelight green solids were suspended in 100 ml 6N HCl and heated at 125° C.under a reflux condenser for 1 hour with intermittent additions of concHCl (2 ml every 15 min). After cooling to RT, 4.38 g (90%) of6-chloro-2,3,4,9-tetrahydro-1H-β-carboline, HCl salt as blue-grey solidwas isolated by filtration.

¹H-NMR (300 MHz, DMSO-d₆): δ 11.33 (br, 2H), 9.62 (br, 2H), 7.53 (d, 1),7.39 (d, 1), 7.09 (dd, 1), 4.33 (br, 2H), 2.92 (t, 2)

Formic Acid Standard Conditions.

DAD RT=1.56 min.

M+H=207.

INTERMEDIATE 38:(6-chloro-1,3,4,9-tetrahydro-g-carbolin-2-yl)-phenyl-methanone

6-chloro-2,3,4,9-tetrahydro-1H-β-carboline, HCl salt (10.2 g, 42 mmol, 1equiv.) was suspended in 100 ml of dry pyridine under N2 and cooled to0° C. in an ice water bath. Benzoyl chloride (7.3 ml, 63 mmol, 1.5equiv.) was added drop-wise to the cold solution after which thereaction was removed from the ice bath and allowed to stir overnight atroom temperature. The reaction was quenched by the addition of wateruntil choked with solids. These solids were captured by filtration,washed with a saturated aqueous sodium bicarbonate solution,re-suspended in water, sonicated, and refiltered to give 1.27 g (94%) of(6-chloro-1,3,4,9-tetrahydro-β-carbolin-2-yl)-phenyl-methanone ascrystalline orange solids.

¹H-NMR (300 MHz, DMSO-d₆): δ 10.65-10.59 (br, 1H), 7.0-7.5 (m, 9H),4.60-4.83 (br, 2H), 3.62-3.99 (br, 2H), 2.75 (br., 2H).

Formic Acid Standard Conditions.

DAD RT=2.68 min.

M+H=311.

INTERMEDIATE 39: 2-benzoyl-6-Chloro-1,2,3,9-tetrahydro-β-carbolin-4-one

(6-chloro-1,3,4,9-tetrahydro-β-carbolin-2-yl)-phenyl-methanone (1.76 g,5.66 mmol, 1 equiv.) and DDQ (2.31 g, 10.2 mmol, 1.8 equiv.) were mixedas solids and cooled to −78° C. 15 ml of a 9:1 THF/H₂O solution wascooled to −78° C. and the resulting slurry was added to the cooledsolids followed by an additional 15 ml of THF (also cooled to −78° C.).The deep blue solution was stirred at −78° C. for 2 hours and thengradually warmed to room temperature and stirred an additional twohours. The reaction was quenched by the addition of 1 N NaOH, andextracted with 3×150 ml EtOAc. The combined organic layers were washedwith 2×100 ml 1 N HCl, 1×100 ml brine, dried over MgSO₄, filtered andconcentrated to yield 1.38 g (75%) of2-benzoyl-6-chloro-1,2,3,9-tetrahydro-β-carbolin-4-one as oily orangesolids.

¹H-NMR (300 MHz, DMSO-d₆): δ 12.11-12.48 (br, 1H), 7.29-7.88 (m, 8H),4.93-5.18 (br, 2H), 4.60-4.46 (br, 2H).

Exact Mass: 324.07.

Formic Acid Standard Conditions

DAD RT=2.15 min.

MPH=325.

INTERMEDIATE 40: 4-amino-6-chloro β-carboline

Crude 2-benzoyl-6-chloro-1,2,3,9-tetrahydro-β-carbolin-4-one (4 g) wasdissolved in 30 ml of anhydrous hydrazine and stirred at reflux (130° C.oil bath) under N₂ for 6 hours, after which the reaction mixture wasallowed to cool to room temperature and sit overnight. The precipitatedyellow solids were removed by filtration and washed with water, 2×5 ml,to yield 785 mg (30%) of 4-amino-6-chloro β-carboline as an off whitesolid. Additional water was added to the combined filtrates until nofurther precipitation occurred. These solids were also removed byfiltration to give 1.056 g (39%) of 4-amino-6-chloro β-carboline asyellow solids, (69% total yield).

¹H-NMR (300 MHz, DMSO-d₆): δ 11.48 (s, 1H), 8.44 (s, 1H), 8.13 (s, 1H),7.77 (s, 1H), 7.42-7.52 (m, 2H), 5.86 (s, 2H).

Formic Acid Standard Conditions.

DAD RT=1.68 min.

M+H=218.

INTERMEDIATE 41:N-(6-chloro-9H-β-carbolin-4-yl)-2,2,2-trifluoro-acetamide

4-amino-6-chloro β-carboline (1.05 g, 4.82 mmol, 1 equiv.) was dissolvedin 4 ml of anhydrous pyridine and 20 ml of THF and cooled to 0° C. underN₂. Trifluoroacetic anhydride (3.4 ml, 24 mmol, 5 equiv.) was addeddrop-wise to the cooled solution. Upon complete addition, the reactionwas removed from the ice bath and stirred at room temperature for ˜1.5hours. The reaction was quenched by the slow addition of water (10 ml)and extracted 2×150 ml EtOAc, washed 2×100 ml saturated aqueous sodiumbicarbonate, 1×100 ml brine, dried over MgSO₄, filtered and concentratedto orange solids. These solids were titurated by in 10-15 ml Et₂O andcaptured by filtration to give 1.23 g (81%) ofN-(6-chloro-9H-β-carbolin-4-yl)-2,2,2-trifluoro-acetamide as a yellowsolid.

¹H-NMR (300 MHz, DMSO-d₆): δ 12.11 (s, 1H), 11.89 (s, 1H), 8.92 (s, 1H),8.33 (s, 1H), 7.82 (s, 1H), 7.60-7.70 (m, 2H).

Formic Acid Standard Conditions.

DAD RT=2.12 min.

M+H=314.

INTERMEDIATE 42:N-(6-chloro-8-nitro-9H-β-carbolin-4-yl)-2,2,2-trifluoro-acetamide

N-(6-chloro-9H-β-carbolin-4-yl)-2,2,2-trifluoro-acetamide (125 mg, 0.4mmol, 1 equiv.) was dissolved in 2 ml TFA and NaNO₂ (541 mg, 7.84 mmol,2 equiv.) was added in one portion. The solution was stirred at roomtemperature for 4 hr. Volatiles were removed by rotovap, and theresulting oily orange solids were suspended in water, neutralized with asaturated aqueous sodium bicarbonate solution and filtered to give 132mg (92%) ofN-(6-chloro-8-nitro-9H-β-carbolin-4-yl)-2,2,2-trifluoro-acetamide.

¹H-NMR (300 MHz, DMSO-d₆): δ 12.87 (s, 1H), 12.03 (s, 1H), 9.11 (s, 1H),8.56 (s, 1H), 8.53 (s, 1H), 8.26 (s, 1H).

Formic Acid Standard Conditions.

DAD RT=2.27 min.

M+H=359.

INTERMEDIATE 43:N-(8-amino-6-chloro-9H-β-carbolin-4-yl)-2,2,2-trifluoro-acetamide

The crudeN-(6-chloro-8-nitro-9H-β-carbolin-4-yl)-2,2,2-trifluoro-acetamide (130mg, 0.36 mmol) was dissolved in 7 ml of MeOH and the reaction vessel wasvacuum purged 3× with N₂. Platinum (20 mg, 20% wt. on activated carbon)was added quickly, and the reaction vessel was again vacuum purged 3×with N₂, followed by 3 additional vacuum purge cycles with H₂. Thereaction was allowed to stir under H₂ at 1 atm overnight. Uponcompletion, the reaction vessel was purged of H₂ and filtered overcelite. The celite was washed several times with methanol until thefiltrates were clear. The combined filtrates were concentrated to giveN-(8-amino-6-chloro-9H-β-carbolin-4-yl)-2,2,2-trifluoro-acetamide as ayellow solid (112 mg, 95%).

¹H-NMR (300 MHz, DMSO-d₆): δ 11.83 (s, 1H), 8.98 (s, 1H), 8.30 (s, 1H),7.10 (s, 1H), 6.82 (s, 1H), 5.87 (br, 2H).

Formic Acid Standard Conditions.

DAD RT=1.95 min.

M+H=329.

INTERMEDIATE 44:N-[6-chloro-4-(2,2,2-trifluoro-acetylamino)-9H-β-carbolin-8-yl]-2-methyl-nicotinamide

N-(8-amino-6-chloro-9H-β-carbolin-4-yl)-2,2,2-trifluoro-acetamide (90mg, 0.274 mmol, 1 equiv.) and 2-methylnicotinic acid (45 mg, 0.329 mmol,1.2 equiv.) were dissolved in 1.5 ml of anhydrous pyridine under N₂.EDCI (84 mg, 0.438 mmol, 1.6 equiv.) was added in one portion and thereaction mixture was stirred at room temperature for 2 hours. Thereaction was quenched with water and the resulting dark solids capturedby filtration. These solids were titurated in a 3:1 methanol-DMSOsolution to giveN-[6-chloro-4-(2,2,2-trifluoro-acetylamino)-9H-β-carbolin-8-yl]-2-methyl-nicotinamideas light yellow solids (41 mg, 3%

¹H-NMR (300 MHz, DMSO-d₆): δ 11.94 (br, 1H), 11.78 (s, 1H), 10.61 (s,1), 9.00 (s, 1), 8.64 (d, 1), 8.38 (s, 1H), 8.13 (d, 1), 8.01 (s, 1H),7.73 (s, 1H), 7.45 (m, 1H), 2.68 (s, 3H).

Formic Acid Standard Conditions.

DAD RT=1.98 min.

M+H=448.

INTERMEDIATE 45:N-[6-chloro-4-(2,2,2-trifluoro-acetylamino)-9H-β-carbolin-8-yl]-nicotinamide

N-(8-amino-6-chloro-9H-β-carbolin-4-yl)-2,2,2-trifluoro-acetamide (90mg, 0.274 mmol, 1 equiv.) and 2-methylnicotinic acid (40 mg, 0.329 mmol,1.2 equiv.) were dissolved in 1.5 ml of anhydrous pyridine under N2.EDCI (84 mg, 0.438 mmol, 1.6 equiv.) was added in one portion and thereaction mixture was stirred at room temperature 2 hours. The reactionwas quenched and treated following the preceding protocol to obtain 38mg (32%) ofN-[6-chloro-4-(2,2,2-trifluoro-acetylamino)-9H-β-carbolin-8-yl]-nicotinamide.

¹H-NMR (300 MHz, DMSO-d₆): δ 11.89 (br, 1H), 10.73 (s, 1), 9.28 (s, 1),8.97 (s, 1), 8.84 (d, 1), 8.43 (d, 1), 8.37 (s, 1), 7.85 (s, 1), 7.76(s, 1), 7.67 (m, 1).

Formic Acid Standard Conditions.

DAD RT=1.94 min.

M+H=434.

Example 28 N-(4-amino-6-chloro-9H-β-carbolin-8-yl)-2-methyl-nicotinamide

N-[6-chloro-4-(2,2,2-trifluoro-acetylamino)-9H-β-carbolin-8-yl]-nicotinamide(41 mg, 0.092 mmol, 1 equiv.) was suspended in 5 ml of MeOH and a 2 mlaqueous solution of K₂CO₃ (127 mg, 0.92 mmol, 10 equiv.) was addedthereto. The resulting clear solution was heated at 60° C. for 16 hr andthen allowed to cool to RT. Additional water was added producing finesolids that were captured by filtration, washed once with 10 ml of 5%MeOH in Et₂O, and dried under high vacuum to give 18 mg ofN-(4-amino-6-chloro-9H-β-carbolin-8-yl)-2-methyl-nicotinamide as powderyyellow solids (56% yield).

¹H-NMR (300 MHz, DMSO-d₆): δ 11.18 (s, 1H), 10.41 (s, 1H), 8.62 (d,J=3.6, 1H), 8.32 (s, 1H), 8.20 (s, 1H), 8.11 (d, J=7.5, 1H), 7.91 (s,1H), 7.79 (s, 1H), 7.43 (m, 1H), 5.91 (br, 2H), 2.66 (s, 3H).

Formic Acid Standard Conditions.

DAD RT=1.63 min.

M+H=352.

Example 29 N-(4-amino-6-chloro-9H-β-carbolin-8-yl)-nicotinamide

N-[6-chloro-4-(2,2,2-trifluoro-acetylamino)-9H-β-carbolin-8-yl]-nicotinamide(38 mg, 0.088 mmol, 1 equiv.) was suspended in 5 ml of MeOH and a 2 mlaqueous solution of K₂CO₃ (121 mg, 0.92 mmol, 10 equiv.) was addedthereto. The resulting clear solution was heated at 60° C. for 11 hr.After cooling to RT, fine solids precipitated that were captured byfiltration and washed with 10 ml of water to give 2.78 mg (10%) ofN-(4-amino-6-chloro-9H-β-carbolin-8-yl)-nicotinamide.

¹H-NMR (300 MHz, DMSO-d₆): δ 11.32 (s, 1H), 10.56 (s, 1H), 9.26 (s, 1H),8.82 (d, 1), 8.42 (d, 1), 8.36 (s, 1H), 8.18 (s, 1H), 7.79 (s, 1H), 7.70(s, 1H), 7.64 (m, 1H), 5.91 (br, 2H).

Formic Acid Standard Conditions.

DAD RT=1.55 min.

M+H=338.

INTERMEDIATE 46: 3-cyanomethyl-indole-1-carboxylic acid tert-butyl ester

A solution of 3-indoleacetonitrile (10 g, 64 mmol) in DMF (160 ml) wasstirred at RT. K₂CO₃ (13.3 g, 96 mmol) and di-tert-butyl dicarbonate(15.35 g, 70 mmol) were added thereto and the reaction mixture wasstirred at RT for 12 hr. H₂O (100 ml) was added to the reaction mixtureand the resulting precipitate was captured by filtration.

The solids were dissolved in hot methanol (20 ml) and the solution wasallowed to cool slowly, producing light orange solids that were isolatedby filtration to give 3-cyanomethyl-indole-1-carboxylic acid tert-butylester (9.2 g).

¹H-NMR (300 MHz, DMSO-d₆): δ 8.08, (d, 1); 7.70-7.66 (m, 2); 7.42-7.29(m, 2); 4.12 (s, 2); 1.63 (3, 9).

NH₄OAc standard conditions.

DAD R_(f)=3.31 min.

M+H=257.

INTERMEDIATE 47: 3-(cyano-methyl-methyl)-indole-1-carboxylic acidtert-butyl ester

A stirred solution of 3-cyanomethyl-indole-1-carboxylic acid tert-butylester (2.15 g, 8.39 mmol) in THF (40 ml) was cooled to −78° C. under anargon atmosphere. Sodium bis(trimethylsilyl)amide (1 M in THF, 10 ml, 10mmol) was added thereto and the cold solution was stirred for 30minutes. Iodomethane (627 uL, 10 mmol) was added thereto and thereaction mixture was stirred 1.5 hr while gradually warming to 0° C. H₂O(100 ml) was added thereto and the solution was brought to RT anddiluted with EtOAc (250 ml). The aqueous layer was removed and extractedwith EtOAc (250 ml). The combined organic layers were washed withaqueous HCl (1N, 3×50 ml), followed by brine, then dried over MgSO₄,filtered, and concentrated to an orange oil. Purification via columnchromatography (hexanes:ethyl acetate) gave3-(cyano-methyl-methyl)-indole-1-carboxylic acid tert-butyl ester (1.8g).

¹H-NMR (300 MHz, DMSO-d₆): δ 8.09 (d, 1); 7.73 (d, 1); 7.69 (s, 1);7.74-7.29 (m, 2); 4.56 (q, 1); 1.66 (d, J=7.2, 3); 1.63 (s, 9).

INTERMEDIATE 48: 3-(2-amino-1-methyl-methyl)-indole-1-carboxylic acidtert-butyl ester

A solution of 3-(cyano-methyl-methyl)-indole-1-carboxylic acidtert-butyl ester (850 mg, 3.14 mol) in methanol (15 ml) was stirred atRT. A Raney-Nickel catalyst (50% g/wt suspension in H₂O, 1 ml) was addedthereto and the reaction vessel was capped and vacuum purged 3 timeswith argon, followed similarly by hydrogen. The mixture was stirred 22hr under 1 atm. of hydrogen, vacuum purged with argon and filteredthrough celite. The filtrate was concentrated to an oil (670 mg) anddetermined by LCMS to be composed mainly of the desired compound3-(2-amino-1-methyl-methyl)-indole-1-carboxylic acid tert-butyl ester.

NH₄OAc standard conditions.

DAD R_(f)=1.78 min.

M+H=275.

INTERMEDIATE 49: 4-methyl-2,3,4,9-tetrahydro-1H-β-carboline-1-carboxylicacid

Crude 3-(2-amino-1-methyl-methyl)-indole-1-carboxylic acid tert-butylester (670 mg, approx. 2.44 mmol) was dissolved in trifluoroacetic acid(2 ml) and stirred 30 min at RT, then concentrated to an oily solidunder reduced pressure. The resulting oil was dissolved in a 3 M ofNaOAc:AcOH buffer solution (pH=4.8, 12 ml) and H₂O (6 ml) at RT.Glyoxalic acid (225 mg, 2.44 mmol) was added thereto and the reactionmixture was stirred for 4 hr at RT, then concentrated to dryness. Theresulting solids were determined to be composed mainly of the desiredcompound 4-methyl-2,3,4,9-tetrahydro-1H-β-carboline-1-carboxylic acid byLCMS and used subsequently thereafter without purification.

NH₄OAc standard conditions.

DAD R_(f)=1.24 min.

M+H=231.

INTERMEDIATE 50: 4-methyl-2,3,4,9-tetrahydro-1H-β-carboline

Crude 4-methyl-2,3,4,9-tetrahydro-1H-β-carboline-1-carboxylic acid(approx. 2.44 mmol) was suspended in H₂O (5 ml) and HCl (12 N, 5 ml) andthe suspension was heated at 120° C. for 1 hr, then allowed to cool toRT. Dark orange-brown solids were removed by filtration and thendissolved in methanol (5 ml). A saturated sodium bicarbonate solution(20 ml) was added thereto, producing a thick yellow slurry. Thisreaction mixture was filtered to yield yellow solids composed mainly of4-methyl-2,3,4,9-tetrahydro-1H-β-carboline (386 mg) as determined byLCMS.

NH₄OAc standard conditions.

DAD R_(f)=1.23 min.

M+H=187.

INTERMEDIATE 51: 4-Methyl-9H-β-carboline

4-methyl-2,3,4,9-tetrahydro-1H-β-carboline (214 mg, 1.17 mmol) wassuspended in xylenes (10 ml). Pd (10% wt. on carbon, 21 mg) catalyst wasadded thereto and the reaction mixture was stirred at 160° C. for 24hours, then cooled to RT and filtered through celite. The filtrate wasconcentrated to dryness to give 4-methyl-9H-β-carboline (210 mg).

¹H-NMR (300 MHz, DMSO-d₆): δ 11.73 (br s, 1); 8.78 (br s, 1); 8.19 (d,1); 8.13 (s, 1); 7.62 (br s, 1); 7.53 (t, 1); 7.25 (t, 1); 2.78 (s, 3),

NH₄OAc standard conditions.

DAD R_(f)=2.24 min.

M+H=183.

INTERMEDIATE 52: 6-Chloro-4-methyl-9H-β-carboline

4-methyl-9H-β-carboline (97 mg, 0.532 mmol) was dissolved in HCl (1N, 4ml) and stirred at RT. NCS (85 mg, 0.637 mmol) was added thereto and thereaction mixture was stirred for 5 hr. Saturated sodium bicarbonatesolution (20 ml) was added thereto and the reaction mixture wasextracted twice with EtOAc (100 ml). The combined organic layers werewashed with brine, dried over MgSO₄, filtered, and concentrated to give6-chloro-4-methyl-9H-β-carboline (108 mg) as an oil.

¹H-NMR (300 MHz, DMSO-d₆): δ 8.67 (s, 1); 8.19 (s, 1); 8.11 (s, 1);7.57-7.54 (m, 2); 2.82 (s, 3).

NH₄OAc standard conditions.

DAD R_(f)=2.48 min.

M+H=217.

INTERMEDIATE 53: 6-chloro-4-methyl-8-nitro-9H-β-carboline

A solution of 6-chloro-4-methyl-9H-β-carboline (100 mg, 0.462 mmol) intrifluoroacetic acid (10 ml) was stirred at RT. NaNO₃ (106 mg, 1.25mmol) was added thereto and the reaction mixture was stirred 30 min,then concentrated to give an orange residue. The residue was dissolvedin MeOH (5 ml) and neutralized by an addition of a saturated sodiumbicarbonate solution (20 ml) causing the formation of yellow solids thatwere captured by filtration, washed with H₂O (10 ml) and Et₂O (2×10 ml)to give 6-chloro-4-methyl-8-nitro-9H-β-carboline (82 mg).

¹H-NMR (300 MHz, DMSO-d₆): δ 8.90 (s, 1); 8.65 (s, 1); 8.55 (s, 1); 8.29(s, 1); 2.89 (s, 3). NH₄OAc standard conditions.

DAD R_(f)=3.00 min.

M+H=262.

INTERMEDIATE 54: 6-chloro-4-methyl-9H-β-carbolin-8-ylamine

A solution of 6-chloro-4-methyl-8-nitro-9H-β-carboline (80 mg, 0.31mmol) in MeOH (10 ml) was stirred at RT. Platinum (10% wt. on carbon, 24mg) catalyst was added thereto and the reaction vessel was capped andvacuum purged 3 times with argon, followed similarly by hydrogen. Thereaction mixture was stirred 1.5 hr under 1 atm hydrogen, then vacuumpurged with argon, diluted with DCM (10 ml) and filtered through a 0.2uM syringe filter. The filtrate was concentrated to yield6-chloro-4-methyl-9H-β-carbolin-8-ylamine (67 mg) as a light brown oil.

¹H-NMR (300 MHz, DMSO-d₆): δ 9.04 (s, 1); 8.29 (s, 1); 7.74 (d, 1); 7.10(d, 1); 2.98 (s, 3).

NH₄OAc standard conditions.

DAD R_(f)=1.89 min,

M+H=231.

Example 30 N-(6-chloro-4-methyl-9H-β-carbolin-8-yl)-nicotinamide

A solution of 6-chloro-4-methyl-9H-β-carbolin-8-ylamine (43 mg, 0.19mmol) in pyridine (4 ml) was stirred at RT under an argon atmosphere.Nicotinoyl chloride hydrochloride (40 mg, 0.22 mmol) was added theretoand the reaction mixture was stirred for 12 hr. The solution was dilutedwith H₂O (5 ml) and poured into a separatory funnel containing H₂O (5ml) and EtOAc (25 ml). The reaction mixture was shaken and the layerswere separated. The aqueous layer was extracted with EtOAc (2×25 ml).The combined organic layers were washed with a saturated sodiumbicarbonate solution (15 ml), followed by brine, then dried over MgSO₄,filtered, and concentrated to yieldN-(6-chloro-4-methyl-9H-β-carbolin-8-yl)-nicotinamide (5.2 mg) as anorange viscous oil.

¹H-NMR (300 MHz, DMSO-d₆): δ 11.67 (s, 1); 10.70 (s, 1); 9.27 (s, 1);8.83 (s, 2); 8.45 (s, 1); 8.20-8.12 (m, 2); 7.83 (s, 1); 7.66 (s, 1);2.80 (s, 3).

NH₄OAc standard conditions.

DAD R_(f)=1.92 min.

M+H=337.

INTERMEDIATE 55: 1,1-Dioxo-1λ⁶-thiomorpholine-3,4-dicarboxylic acid4-tert-butyl ester

NOM Thiomorpholine-3,4-dicarboxylic acid 4-tert-butyl ester (120 mg,0.485 mmol) was dissolved in Et₂O (8 ml). To the solution was addedmCPBA (172 mg, 0.994 mmol), followed later by a second portion of mCPBA(84 mg, 0.485 mmol). The precipitate which formed was filtered, washedwith Et₂O and dried to yield a white solid of1,1-dioxo-1λ⁶-thiomorpholine-3,4-dicarboxylic acid 4-tert-butyl ester(74 mg).

Example 31 1,1-Dioxo-1λ⁶-thiomorpholine-3-carboxylic acid(6-chloro-9H-β-carbolin-8-yl)-amide

A slurry of 6-chloro-7-fluoro-9H-β-carbolin-8-ylamine (43 mg, 0.198mmol), 1,1-dioxo-1λ⁶-thiomorpholine-3,4-dicarboxylic acid 4-tert-butylester (72 mg, 0.257 mmol), and EDCI (76 mg, 0.396 mmol) in pyridine (2ml) was heated to 70° C. After 1 hr, the solvent was removed underreduced pressure and the resulting dark oil was dissolved in MeOH (1ml). The MeOH solution was added drop-wise to a stirring solution ofaqueous NaHCO₃ and a yellow precipitate was formed. The solid wasfiltered, dried, and dissolved in 2 M HCl in Et₂O. After stirringovernight the resulting yellow solid was filtered and dried to yield ayellow solid, the di-HCl salt of1,1-Dioxo-1λ⁶-thiomorpholine-3-carboxylic acid(6-chloro-9H-β-carbolin-8-yl)-amide (78 mg).

¹H-NMR (300 MHz, MeOH-d₄): δ 9.26 (s, 1); 8.74 (d, 1); 8.54 (d, 1); 8.37(d, 1); 7.94 (d, 1); 5.01 (dd, 1); 4.15-3.75 (m, 4); 3.64-3.58 (m, 2).

NH₄OAc standard conditions.

DAD R_(f)=1.57 min.

M+H=379.

INTERMEDIATE 56: 6,6-Dimethyl-morpholine-3,4-dicarboxylic acid4-tert-butyl ester

To a suspension of 6,6-dimethyl-morpholine-3-carboxylic acid (5.56 g,34.9 mmol) in dioxane (58 mL) was added aqueous potassium carbonate (1M,58 mL). To the resulting clear colorless solution was addeddi-tert-butyl dicarbonate (9.14 g, 41.9 mmol). The solution was stirredat room temperature overnight. The reaction mixture was diluted withwater (200 mL) and the pH of the solution was confirmed to beapproximately 7. The reaction mixture was poured into a separatoryfunnel and extracted with Et₂O (2×100 mL) to remove excess di-tert-butyldicarbonate. The aqueous layer was acidified by addition of 6N aqueousHCl with stirring until a pH of 3 was reached. The mixture was quicklyextracted with Et₂O (2×200 mL) and the organic layers were combined,dried over magnesium sulfate, filtered, and concentrated to yield aclear colorless oil. The oil was dissolved in Et₂O (50 mL), trituratedwith hexanes (150 mL), and concentrated to yield a white solid. Thesolid product was placed on the high-vacuum pump for several hours,after which 8.85 g of 6,6-dimethyl-morpholine-3,4-dicarboxylic acid4-tert-butyl ester was obtained (97% yield).

¹H-NMR (300 MHz, DMSO-d₆): δ 12.95 (s, 1); 4.35 (“dd”, 1); 3.98-3.83 (m,2); 3.48 (“dd”, 1); 2.81 (“dd”, 1); 1.39 (“dd”, 9); 1.15 (s, 3); 1.08(“dd”, 3).

NH₄OAc standard conditions.

DAD R_(f)=0.98 min.

M−H=258.

INTERMEDIATE 57:(S)-5-(6-Chloro-9H-β-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholine-4-carboxylicacid tert-butyl ester

The desired compound was prepared according to Method C from6-chloro-9H-β-carboline-8-ylamine and6,6-dimethyl-morpholine-3,4-dicarboxylic acid 4-tert-butyl ester in 87%yield.

¹H-NMR (DMSO-d₆, 300 MHz) δ 11.33 (s, 1); 10.14 (s, 1); 9.05 (s, 1);8.38 (d, 1); 8.21 (d, 1); 8.16 (d, 1); 7.94 (s, 1); 4.70-4.56 (m, 1);4.25-4.14 (m, 1); 4.07 (dd, 1); 3.64-3.56 (m, 1); 3.30-3.14 (m, 1); 1.41(“dd”, 9); 1.21 (s, 3); 1.15 (s, 3).

NH₄OAc standard conditions.

DAD R_(f)=1.84 min.

M+H=459.

Chiral HPLC: 95% ee.

Chiralpak AD column.

15% v/v EtOH/Hexanes containing 0.1% Et₂NH.

INTERMEDIATE 58:[(S)-4-((S)-2-Amino-propyl)-6,6-dimethyl-morpholine-3-carboxylic acid(6-chloro-9H-beta-carbolin-8-yl)-amide trifluoroacetate salt

The desired compound was prepared using the same procedure as forIntermediate 35 starting from(S)-5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholine-4-carboxylicacid tert-butyl ester and using Boc-alaninal in the reductive alkylationstep in 60% yield.

¹H-NMR (300 MHz, D₂O): δ 1.16 (d, 3H), 1.25 (s, 3H), 1.28 (s, 3H), 2.41(d, 1H), 2.59 (dd, 1H), 2.89 (dd, 1H), 2.95 (d, 1H), 3.35-3.50 (m, 2H),3.95-4.15 (m, 2H), 7.59 (d, 1H), 7.97 (d, 1H), 8.11 (d, 1H), 8.30 (d,1H), 8.40 (d, 1H), 8.94 (s, 1H).

Retention Time (LC, method: ammonium acetate standard): 1.57 min.

MS (M+H⁺): 416.2.

METHOD E: Coupling procedure using[(S)-4-((S)-2-Amino-propyl)-6,6-dimethyl-morpholine-3-carboxylic acid(6-chloro-9H-beta-carbolin-8-yl)-amide trifluoroacetate salt

[(S)-4-((S)-2-Amino-propyl)-6,6-dimethyl-morpholine-3-carboxylic acid(6-chloro-9H-beta-carbolin-8-yl)-amide trifluoroacetate salt (1.0mmole), TBTU (1.2 mmoles), the acid (1.25 mmoles) to be coupled and Et₃N(4-6 mmoles, basic pH) were taken into acetonitrile (10 ml). Theresulting mixture was stirred at ambient temperature for 4-15 hrs. Thereaction mixture was then partitioned into EtOAc and 10% aqueous Na₂CO₃solution. The separated aqueous phase was further extracted with EtOAc.The combined extracts were successively washed with 10% aqueous Na₂CO₃solution and brine, dried over Na₂SO₄ and concentrated completely. Theresidue was purified on silica (2-7% MeOH/CH₂Cl₂) to give thecorresponding product.

Example 396,6-Dimethyl-4-[2-(2,2,2-trifluoro-acetylamino)-propyl]-morpholine-3-carboxylicacid (6-chloro-9H-β-carbolin-8-yl)-amide

4-(2-Amino-propyl)-6,6-dimethyl-morpholine-3-carboxylic acid(6-chloro-9H-β-carbolin-8-yl)-amide (3 CF₃COOH salt) (1.5 g) wassuspended in dichloromethane (80 mL) along with 5 equivalents oftriethylamine. Trifluoroacetic anhydride (56 μL, 2 equivalents) wasadded and the mixture stirred at room temperature for an hour. Thesolvent was removed by rotary evaporation. The product was purified bysilica gel flash chromatography (5% methanol/dichloromethane, product Rf0.3) to afford 1 g.

¹H-NMR (300 MHz, relative to CDCl₃ peak at 7.3 ppm) δ 10 (s, 1H), 9.7(d, 1H), 8.7 (s, 1H), 8.6 (s, 1H), 8.2 (d, 1H), 7.6 (s, 2H), 6.6 (s,1H), 4.3 (m, 1H), 3.9 (m, 1H), 3.8 (t, 1H), 3.2 (m, 1H), 2.7-2.9 (m,2H), 2.5 (m, 1H), 2.2 (d, 1H), 1.4 (d, 3H), 1.3 (s, 3H), 1.2 (s, 3H).

LCMS (ammonium acetate standard method) retention time=1.84 min.

(M⁺=512; M⁻=510).

Example 404-((S)-2-Acetylamino-propyl)-6,6-dimethyl-morpholine-3-(S)-carboxylicacid (6-chloro-9H-β-carbolin-8-yl)-amide

The desired compound was prepared according to the previous example from4-(2-Amino-propyl)-6,6-dimethyl-morpholine-3-carboxylic acid(6-chloro-9H-β-carbolin-8-yl)-amide (3 CF₃COOH salt) and aceticanhydride.

¹H-NMR (300 MHz, methyl-d₃ alcohol-d): δ 1.15 (d, 3H), 1.23 (s, 3H),1.39 (s, 3H), 1.98 (s, 3H), 2.24 (d, 1H), 2.38 (m, 1H), 2.68 (m, 1H),2.92 (d, 1H), 3.24 (m, 1H), 3.98 (m, 2H), 4.22 (m, H), 7.78 (d, 1H),7.98 (m, 2H), 8.27 (d, 1H), 8.84 (s, 1H).

Retention Time (LC, method: ammonium acetate standard): 1.54 min.

MS (M+H⁺): 458.

Example 414-((8)-2-Methanesulfonylamino-propyl)-6,6-dimethyl-morpholine-3-(S)-carboxylicacid (6-chloro-9H-β-carbolin-8-yl)-amide

The desired compound was prepared according to Method E.

¹H-NMR (300 MHz, methyl-d₃ alcohol-d): δ 1.28 (s, 3H), 1.29 (d, 3H),1.43 (s, 3H), 2.28 (d, 1H), 2.57 (m, 1H), 2.66 (m, 1H), 2.98 (s, 3H),3.03 (d, 1H), 3.34 (m, 1H), 3.66 (m, 1H), 4.05 (m, 2H), 7.67 (d, 1H),8.10 (m, 2H), 8.32 (d, 1H), 8.89 (s, 1H).

Retention Time (LC, method: ammonium acetate standard): 1.54 min.

MS (M+H⁺): 494.

Example 424-{2-[(-4,6-Dimethyl-pyrimidine-5-carbonyl)-amino]-propyl}-6,6-dimethyl-morpholine-3-carboxylicacid (6-chloro-9H-beta-carbolin-8-yl)-amide

The desired compound was prepared using[4-(2-Amino-propyl)-6,6-dimethyl-morpholin-3-ylmethyl]-(6-chloro-9H-beta-carbolin-8-yl)-amineand 4,6-dimethylpyrimidine-5-carboxylic acid following Method E in 51%yield.

¹H-NMR (300 MHz, DMSO): δ 11.27 (1H, s), 10.02 (1H, s), 9.0 (1H, s),8.86 (1H, s), 8.5 (1H, d), 8.3 (1H, d), 8.22 (2H, m), 7.88 (1H, s), 4.1(1H, m), 3.9 (2H, m), 2.99 (2H, m), 2.36 (6H, s), 2.1 (2H, m), 1.3 (3H,s), 1.24 (6H, m).

Retention time (LC, method: ammonium acetate standard): 1.50 min.

MS (M+H⁺): 551

Example 43(S)-6,6-Dimethyl-4-((S)-2-[(2-methyl-pyridine-3-carbonyl)amino]propyl)-morpholine-3-carboxylicacid (6-chloro-9H-beta-carbolin-8-yl)-amide

The desired compound was prepared according to Method E from(S)-4-((S)-2-Amino-propyl)-6,6-dimethyl-morpholine-3-carboxylic acid(6-chloro-9H-beta-carbolin-8-yl)-amide trifluoroacetate salt and2-methyl-nicotinic acid in 75% yield.

¹H-NMR (300 MHz, DMSO-d₆): δ 1.21 (s, 3H), 1.22 (d, 3H), 1.36 (s, 3H),2.10 (d, 1H), 2.42 (m, 1H), 2.60 (m, 1H), 2.99 (d, 1H), 3.20 (m, 1H),3.92 (m, 2H), 4.22 (m, 1H), 7.22 (dd, 1H), 7.65 (d, 1H), 7.90 (s, 1H),8.16 (d, 1H), 8.23 (s, 1H), 8.31 (d, 1H), 8.38 (d, 1H), 8.45 (d, 1H),9.02 (s, 1H), 10.04 (s, 1H), 11.26 (s, 1H).

Retention Time (LC, method: ammonium acetate standard): 2.16 min.

MS (M+H⁺): 535.5.

Example 446,6-Dimethyl-4-{2-[(tetrahydro-pyran-4-carbonyl)-amino]-propyl}-morpholine-3-carboxylicacid (6-chloro-9H-β-carbolin-8-yl)-amide

4-(2-Amino-propyl)-6,6-dimethyl-morpholine-3-carboxylic acid(6-chloro-9H-β-carbolin-8-yl)-amide (3 CF₃COOH salt) (300 mg) wassuspended in methylene chloride (12 mL) along with 3 equivalents oftriethylamine. Morpholine-4-carbonyl chloride (70 mg, 1.3 equivalents)was added and the mixture stirred at room temperature overnight. Thesolvent was removed by rotary evaporation. The product was separated bypreparative TLC on silica plates (10/90 methanol/ethyl acetate aseluent, product Rf 0.4) Yield: 83 mg.

¹H-NMR (300 MHz, relative to CD₃OD peak at 3.3 ppm) δ 8.8 (s, 1H), 8.27(d, 1H), 7.9-7.99 (m, 3H), 3.95-4.15 (m, 2H), 3.85-3.95 (m, 2H), 3.5-3.6(m, 5H), 3.3-3.45 (m, 2H), 3.15-3.3 (m, 2H), 2.85-2.95 (d, 1H), 2.6-2.72m, 1H), 2.3-2.43 (m, 1H), 2.2-2.28 (d, 1H), 2.0 (s, 2H), 1.35-1.45 (d,3H), 1.05-1.25 (m, 6H).

LCMS (ammonium acetate standard method) retention time=2.39 min.(M+=529; M−=527).

Example 454-{(S)-2-[(1-Acetyl-pyrrolidine-2-(S)-carbonyl)-amino]-propyl}-6,6-dimethyl-morpholine-3-(S)-carboxylicacid (6-chloro-9H-β-carbolin-8-yl)-amide

The desired compound was prepared according to Method E.

¹H-NMR (300 MHz, methyl-d₃ alcohol-d): δ 1.21 (d, 3H), 1.28 (s, 3H),1.39 (s, 3H), 1.95 (m, 3H), 2.05 (s, 3H), 2.17 (m, 1H), 2.28 (d, 1H),2.51 (m, 1H), 2.74 (m, 1H), 3.05 (d, 1H), 3.30 (m, 1H), 3.55 (m, 1H),3.61 (m, 1H), 4.05 (m, 2H), 4.18 (m, 1H), 4.34 (m 1H), 7.78 (d, 1H),8.10 (m, 2H), 8.33 (d, 1H), 8.90 (s, 1H).

Retention Time (LC, method: ammonium acetate standard): 2.07 min.

MS (M+H⁺): 555.

Example 466,6-Dimethyl-4-{-2-[(5-methyl-isoxazole-3-carbonyl)-amino]-propyl}-morpholine-3-carboxylicacid (6-chloro-9H-beta-carbolin-8-yl)-amide

The desired compound was prepared using[4-(2-Amino-propyl)-6,6-dimethyl-morpholin-3-ylmethyl]-(6-chloro-9H-beta-carbolin-8-yl)-amineand 5-methylisoxazole carbonyl chloride following Method E in 61% yield.

¹H-NMR (300 MHz, DMSO): δ 11.2 (1H, s), 9.98 (1H, s), 9.0 (1H, s), 8.7(1H, d), 8.6 (1H, d), 8.2 (2H, m), 7.9 (1H, s). 6.47 (1H, s), 3.87 (2H,m), 3.17 (2H, m), 2.9 (1H, d), 2.7 (1H, m), 2.3 (4H), 2.1 (1H, d), 1.29(3H, s), 1.15 (6H, m).

Retention time (LC, method: ammonium acetate standard): 1.81 min.

MS (M+H+): 526

Example 476,6-Dimethyl-4-[2-(3-methyl-ureido)-prop1yl]-morpholine-3-carboxylicacid (6-chloro-9H-β-carbolin-8-yl)-amide

4-(2-Amino-propyl)-6,6-dimethyl-morpholine-3-carboxylic acid(6-chloro-9H-□-carbolin-8yl)-amide (3 HCl salt) (300 mg) was suspendedin dichloromethane (10 mL) triethyl amine (4 equivalents) and methylisocyanate (2 equivalents) were added simultaneously. After one hour atroom temperature solvent was removed by rotary evaporation. The productwas purified by silica gel flash chromatography (5%methanol/dichloromethane, product Rf=0.3) to afford 200 mg.

¹H-NMR (300 MHz, relative to CDCl₃ peak at 7.3 ppm) δ 12.3 (s, 1H), 10.2(s, 1H), 8.9 (s, 1H), 8.6 (s, 1H), 8.4 (d, 1H), 7.9 (d, 1H), 7.8 (s,1H), 5.4 (s, 1H), 5.2 (d, 1H), 4.2 (s, 1H), 3.8 (m, 2H), 3.2 (m, 1H),2.6-3 (m, 7H), 2.3 (d, 1H), 2.2 (t, 1H), 1.4 (s, 3H), 1.2 (s, 3H), 1.1(d, 3H)

LCMS (ammonium acetate standard method) retention time=1.62 min.(M⁺=473; M⁻=471).

Example 49{(S)-2-[(S)-5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-yl]-1-methyl-ethyl}-carbamicacid methyl ester

To a solution of (S)-4-((S)-2-Amino-propyl)-6,6-dimethyl-morpholine-3carboxylic acid (6-chloro-9H-beta-carbolin-8-yl)-amide hydrochloridesalt (3.45 g, 6.59 mole) in 68 ml of dry pyridine, was added in threeportions over 1.5 hr, a 3M DCM solution of methyl chloroformate (9.2 ml,27.6 mole, 4.2 eq). After 2 h, 10 ml of water were added and the mixturewas concentrated to dryness. The residue was partitioned into 150 ml 0fEtOAc and 100 ml of an aqueous 0.5M solution of K₂CO₃. The separatedaqueous phase was extracted with 50 ml of EtOAc. The combined organicextracts were successively washed with water (2×50 ml) and brine (50ml), dried over Na₂SO₄ and concentrated to dryness. The residue waspurified on silica (5% MeOH/CH₂Cl₂) to give 2.48 g (thick oil, 77%yield) of the desired product.

¹H-NMR (300 MHz, CDCl₃): δ 1.15 (d, 3H), 1.28 (s, 3H), 1.42 (s, 3H),2.33 (dd, 1H), 2.42 (d, 1H), 2.78 (dd, 1H), 2.86 (d, 1H), 3.32 (dd, 1H),3.86 (s, 3H), 3.92 (t, 1H), 4.01 (dd, 1H), 4.18 (m, 1H), 4.78 (d, 1H),7.95 (d, 1H), 7.97 (s, 1H), 8.29 (s, 1H), 8.50 (d, 1H), 8.98 (s, 1H),9.88 (s, 1H), 10.94 (s, 1H).

Retention Time (LC, method: ammonium acetate standard): 1.70 min.

MS (M+H⁺): 474.1

Example 504-{2-[(2,4-Dimethyl-pyridine-3-carbonyl)-amino]-propyl}-6,6-dimethyl-morpholine-3-carboxylicacid (6-chloro-9H-beta-carbolin-8-yl)-amide

The desired compound was prepared from[4-(2-Amino-propyl)-6,6-dimethyl-morpholin-3-ylmethyl]-(6-chloro-9H-beta-carbolin-8-yl)-amineand 2,4-dimethyl nicotinic acid following Method E in 50% yield.

¹H-NMR (300 MHz, DMSO): δ 11.27 (1H, s), 10 (1H, s), 8.9 (1H, s), 8.37(2H, d), 8.24 (3H, m), 7.8 (1H, s), 7.04 (1H, d), 3.91 (2H, m), 3.1 (2H,m), 2.36 (4H, m), 2.1 (3H, m), 2.05 (1H, d), 1.3 (3H, s), 1.2 (6H, m).

Retention time (LC, method: ammonium acetate standard): 1.53 min.

MS (M+H⁺): 550

Example 516,6-dimethyl-4-(2-[(pyrazine-2-carbonyl)-amino]-propyl)-morpholine-3-carboxylicacid (6-chloro-9H-beta-carbolin-8-yl)-amide

The desired compound was prepared from[4-(2-Amino-propyl)-6,6-dimethyl-morpholin-3-ylmethyl]-(6-chloro-9H-beta-carbolin-8-yl)-amineand 2-pyrazine carboxylic acid following Method E in 62% yield.

¹H-NMR (300 MHz, DMSO): δ 12.9 (1H, s), 10.89 (1H, s), 9.42 (1H, s), 9.0(1H, s), 8.8 (1H, d), 8.6 (1H, d), 8.4 (1H, s), 8.2 (1H, m), 4.5 (1H,m), 4.1 (2H, m), 3.1 (1H, m), 2.1 (4H, m), 1.23 (9H, m).

Retention time (LC, method: ammonium acetate standard): 1.69 min.

MS (M+): 522.2

Example 52 Pyridine-3,4-dicarboxylic acid4-({2-[5-(6-chloro-9H-β-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-yl)-1-methyl-ethyl]-amide}3-methylamide

To a solution of[4-(2-Amino-propyl)-6,6-dimethyl-morpholin-3-ylmethyl]-(6-chloro-9H-beta-carbolin-8-yl)-amine(100 mg, 0.132 mmole) in 0.6 ml of dry acetonitrile, was added3,4-pyridinedicarboxylic anhydride (21 mg, 0.15 mmole) and triethylamine(102 ml, 0.8 mmole). The reaction mixture was stirred at ambienttemperature for 1 h. The solvent was then removed under reduced pressureand the residue was taken up into pyridine (0.6 ml). To the resultingmixture was added 2M methylamine solution in THF (0.2 ml, 0.4 mmole) andEDCI (40 mg, 0.21 mmole). The reaction mixture was stirred for 4 hrs,the solvent was removed under reduce pressure and the residue waspartitioned into EtOAc and 1M aqueous K₂CO₃. The separated aqueous phasewas extracted twice with EtOAc. The combined organic phases weresuccessively washed with water and brine, dried over MgSO₄, andconcentrated completely. The residue was purified on silica gel (10%MeOH—CH₂Cl₂) to give the title compound as a white solid in 36% yield.

¹H-NMR (300 MHz, DMSO): δ 9.24 (1H, s), 8.17 (1H, s), 7.81 (2H, m), 7.5(3H, m), 7.3 (1H, s), 7.0 (1H, s) 6.5 (1H, d), 3.08 (2H, d), 3.35 (1H,m), 2.4 (3H, m), 1.86 (4H, m), 0.5 (3H, s), 0.37 (6H, m).

Retention time (LC, method: ammonium acetate standard): 1.46 min.

MS (M+H⁺): 579

Example 536.6-Dimethyl-4-{(4-methyl-pyrimidine-5-carbonyl)-amino)-propyl}-morpholine-3-carboxylicacid (6-chloro-9H-beta-carbolin-8-yl)-amide

The desired compound was prepared according to Method E from[4-(2-Amino-propyl)-6,6-dimethyl-morpholin-3-ylmethyl]-(6-chloro-9H-beta-carbolin-8-yl)-amineand 4-methyl-pyrimidine-5-carboxylic acid in 55% yield.

¹H-NMR (300 MHz, DMSO): δ 11.26 (1H, s), 10.04 (1H, s) 9.04 (2H, m),8.66 (1H, d) 8.3 (2H, m) 8.22-8.17 (2H, m), 7.9 (1H, s), 4.2 (1H, m),3.93 (2H, m), 3.2 (1H, m), 2.98 (2H, m), 2.6 (3H, m), 2.1 (2H, m), 1.36(3H, s), 1.23 (6H, m).

Retention time (LC, method: ammonium acetate standard): 1.55 min.

MS (M+H+): 537

Example 54(S)-6,6-Dimethyl-4-{(S)-2-[(4-methyl-pyridine-3-carbonyl)-amino]-propyl}-morpholine-3-carboxylicacid (6-chloro-9H-beta-carbolin-8-yl)-amide

The desired compound was prepared according to Method E from(8)-4-((S)-2-Amino-propyl)-6,6-dimethyl-morpholine-3-carboxylic acid(6-chloro-9H-beta-carbolin-8-yl)-amide trifluoroacetate salt and4-methyl-nicotinic acid in 79% yield.

¹H-NMR (300 MHz, DMSO-d₆): δ 1.21 (s, 3H), 1.22 (d, 3H), 1.36 (s, 3H),2.10 (d, 1H), 2.40 (m, 1H), 2.62 (m, 1H), 2.99 (d, 1H), 3.22 (m, 1H),3.94 (m, 2H), 4.23 (m, 1H), 7.26 (d, 1H), 7.90 (s, 1H), 8.16 (d, 1H),8.23 (s, 1H), 8.34-8.46 (m, 3H), 9.02 (s, 1H), 10.04 (s, 1H), 11.27 (s,1H).

Retention Time (LC, method: ammonium acetate standard): 2.22 min.

MS (M+H⁺): 535.5.

Example 474-[2-(2-Amino-2-methyl-propionyl)]-6,6-dimethyl-morpholine-3-carboxylicacid (6-chloro-9H-β-carbolin-8-yl)-amide

A solution of {2-[5-(6-Chloro-9H-β-carbolin-8-ylcarbamoyl)2,2-dimethyl-morpholin-4-yl]-1-methyl-ethyl}-carbamic acid tert-butylester (70.2 mg, 0.14 mmol) in TFA (2 mL) was stirred at roomtemperature. After 15 min, the reaction was concentrated and the crudeproduct was azeotroped with CH₂Cl₂ (2×5 mL). A mixture of the crudeintermediate, TBTU (54.0 mg, 0.17 mmol), triethylamine (0.2 mL, 1.43mmol) and 2-tert-butoxycarbonylamino-2-methyl-propionic acid (45.0 mg,0.22 mmol) in MeCN (1 mL) was stirred at room temperature for 18 h. Thesolution was diluted with H₂O (20 mL) and poured into a separatoryfunnel containing EtOAc (50 mL), and brine (50 mL). The mixture wasshaken and the layers were separated. The aqueous layer was extractedwith EtOAc (2×50 mL). The combined organic layers were dried, filteredand concentrated. The crude product was purified by flash chromatographyto yield a yellow solid (51.0 mg, 62%) which was shown by NMR and LCMSto be4-[2-(2-amino-2-methyl-propionyl]-6,6-dimethyl-morpholine-3-carboxylicacid (6-chloro-9H-β-carbolin-8-yl)-amide.

¹H-NMR (300 MHz, DMSO-d₆): δ 12.94 (br s, 1); 11.36 (br s, 1); 10.16 (s,1); 9.06 (s, 1); 8.39 (d, 1); 8.22 (d, 1); 8.19 (d, 1); 7.95 (s, 1);4.77-4.52 (m, 1); 4.28-4.13 (m, 1); 4.13-4.00 (m, 1); 3.68-3.52 (m, 1);3.22-3.12 (m, 1); 1.44 (s, 3); 1.41-1.38 (m, 6); 1.28-1.24 (m, 2); 1.22(s, 3); 1.25 (s, 3); 1.11-1.07 (m, 1).

NH₄OAc standard conditions.

DAD R_(f)=1.31 min.

M+H=501.

Example 55 6,6Dimethyl-4-(1,2,3,4-tetrahydroisoquinolin-3-ylmethyl)-morpholine-3-carboxylicacid (6-chloro-9H-beta-carbolin-8-yl)amide

The desired compound was made following the procedure outlined in MethodC using 6,6-dimethyl morpholine-3-carboxylic acid and(s)-tetrahydroisoquinoline aldehyde.

¹H-NMR (300 MHz, D₂O): δ 9.1 (1H, s), 8.68 (1H, d), 8.52 (1H, d), 8.41(1H, d), 7.68 (1H, d), 7.27 (1H, d), 7.06 (1H, m), 6.97 (1H, d), 6.84(1H, m), 4.31 (2H, m), 4.09 (2H, m), 3.68 (1H, m), 3.56 (1H, t), 3.2(2H, m), 3.06 (2H, m), 2.7 (2H, m) 1.48 (3H, s), 1.32 (3H, s)

Retention time (LC, method: ammonium acetate standard): 2.43 min.

MS (M+H⁺): 505

INTERMEDIATE 59: 6,6-Dimethyl-morpholine-3-carboxylic acid(6-chloro-9H-β-carbolin-8-yl)-amide, HCl salt

To a clear brown solution of5-(6-chloro-9H-(3-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholine-4-carboxylicacid tert-butyl ester (10.4 g, 22.7 mmol) in methanol (41 mL) was addedHCl in dioxane (4M, 91 mL). The reaction was stirred for 30 minutes atroom temperature, during which time a pale brown precipitate began toform. The mixture was poured into a 250-mL volume of vigorously stirringEt₂O. The resulting slurry was stirred at room temperature for 15minutes, then filtered to yield a pale orange solid. The solid wasplaced on the high-vacuum pump overnight, after which 9.71 g of6,6-dimethyl-morpholine-3-carboxylic acid(6-chloro-9H-β-carbolin-8-yl)-amide was obtained (99% yield).

¹H-NMR (DMSO-d₆, 300 MHz) δ 13.47 (s, 1); 11.77 (s, 1); 9.42 (s, 1);8.86 (d, 1); 8.66 (d, 1); 8.58 (d, 1); 8.25 (d, 1); 4.41-4.37 (m, 2);4.05 (dd, 1); 3.32-3.28 (m, 1); 3.04-3.00 (m, 1); 1.34 (s, 3); 1.30 (s,3).

NH₄OAc standard conditions.

DAD R_(f)=1.48 min.

M+H=359.

Example 56 4-(2-Amino-butyl)-6,6-dimethyl-morpholine-3-carboxylic acid(6-chloro-9H-β-carbolin-8-yl)-amide (also INTERMEDIATE 60)

Method C was followed, using5-(6-chloro-9H-β-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholine-4-carboxylicacid tert-butyl ester and the appropriate aldehyde,(1-formyl-propyl)-carbamic acid tert-butyl ester.

¹H-NMR (DMSO-d₆, 300 MHz) δ 9.06 (s, 1); 8.37 (d, 1); 8.19 (d, 1); 8.15(d, 1); 7.85 (d, 1); 6.75 (br s, 2); 3.96-3.85 (m, 2); 3.17-3.13 (m, 1);2.89 (d, 1); 2.78-2.74 (m, 1); 2.67-2.59 (m, 1); 2.26-2.20 (m, 1); 2.14(d, 1); 1.58-1.50 (m, 1); 1.32 (s, 3); 1.32-1.23 (m, 1); 1.18 (s, 3);0.87 (t, 3).

NH₄OAc standard conditions.

DAD R_(f)=1.27 min.

M+H=430.

Example 576,6-Dimethyl-4-(2-[(2-methyl-pyridine-3-carbonyl)-amino]-butyl)-morpholine-3-carboxylicacid (6-chloro-9H-β-carbolin-8-yl)-amide, HCl salt

To a solution of 4-(2-amino-butyl)-6,6-dimethyl-morpholine-3-carboxylicacid (6-chloro-9H-β-carbolin-8-yl)-amide (100 mg, 0.233 mmol) inpyridine (4 mL) was added 2-methyl-nicotinic acid (38.4 mg, 0.280 mmol)and EDCI (71.5 mg, 0.373 mmol). The solution was stirred overnight atroom temperature, then diluted with water (5 mL). The mixture was pouredinto a separatory funnel and diluted further with water (20 mL). Themixture was extracted with EtOAc (2×20 mL), then the combined organiclayers were washed with brine. The organic layer was dried overmagnesium sulfate, filtered, and concentrated to yield a yellow-brownoil which was purified via column chromatography. The resulting yellowsolid was dissolved in methanol (2 mL) and HCl in Et₂O (2M, 2 mL) wasadded. The mixture was stirred for 5 minutes, then concentrated to yield103 mg of6,6-dimethyl-4-(2-[(2-methyl-pyridine-3-carbonyl)-amino]-butyl)-morpholine-3-carboxylicacid (6-chloro-9H-β-carbolin-8-yl)-amide (71% yield).

¹H-NMR (DMSO-d₆, 300 MHz) δ 13.31 (br s, 1); 11.41 (br s, 1); 11.14 (brs, 1); 9.44 (s, 1); 8.85 (d, 1); 8.73-8.64 (m, 3); 8.52 (s, 1); 8.32 (s,1); 7.72 (s, 1); 4.64-3.55 (m, 6); 3.24-3.06 (m, 1); 2.95-2.81 (m, 1);2.71 (s, 3); 1.89-1.74 (m, 1); 1.55-1.41 (m, 1); 1.32 (s, 3); 1.23 (s,3); 0.93 (t, 3).

NH₄OAc standard conditions.

DAD R_(f)=1.66 min.

M+H=549.

INTERMEDIATE 61:4-(2-Amino-3-methyl-butyl)-6,6-dimethyl-morpholine-3-carboxylic acid(6-chloro-9H-β-carbolin-8-yl)-amide

Method C was followed, using5-(6-chloro-9H-β-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholine-4-carboxylicacid tert-butyl ester and the appropriate aldehyde,(1-formyl-2-methyl-propyl)-carbamic acid tert-butyl ester.

¹H-NMR (DMSO-d₆, 300 MHz) δ 9.05 (s, 1); 8.37 (d, 1); 8.21 (d, 1); 8.16(dd, 1); 7.90 (d, 1); 6.71 (br s, 2); 3.93-3.86 (m, 2); 3.18-3.14 (m,1); 2.91 (d, 1); 2.70-2.65 (m, 2); 2.21 (dd, 1); 2.15 (d, 1); 1.74-1.66(m, 1); 1.31 (s, 3); 1.19 (s, 3); 0.85 (d, 3); 0.80 (d, 3).

NH₄OAc standard conditions.

DAD R_(f)=1.27 min.

M+H=444.

Example 586,6-Dimethyl-4-{3-methyl-2-[(2-methyl-pyridine-3-carbonyl)-amino]-butyl}-morpholine-3-carboxylicacid (6-chloro-9H-β-carbolin-8-yl)-amide

To a solution of4-(2-amino-3-methyl-butyl)-6,6-dimethyl-morpholine-3-carboxylic acid(6-chloro-9H-β-carbolin-8-yl)-amide (1.47 g, 3.31 mmol) in pyridine (35mL) was added 2-methyl-nicotinic acid (544 mg, 3.97 mmol) and EDCI (1.02g, 5.30 mmol). The solution was stirred 6.5 hours at room temperature,then diluted with water (100 mL). The mixture was poured into aseparatory funnel and diluted further with water (50 mL) and EtOAc (150mL). The layers were shaken and separated. The aqueous layer wasextracted with EtOAc (3×50 mL), then the combined organic layers werewashed with brine. The organic layer was dried over magnesium sulfate,filtered, and concentrated to yield an orange semi-solid residue whichwas purified via column chromatography. The resulting yellow solid wasplaced on the high-vacuum pump overnight, after which 1.43 g of6,6-dimethyl-4-{3-methyl-2-[(2-methyl-pyridine-3-carbonyl)-amino]-butyl}-morpholine-3-carboxylicacid (6-chloro-9H-β-carbolin-8-yl)-amide was obtained (77% yield).

¹H-NMR (DMSO-d₆, 300 MHz) δ 11.32 (s, 1); 10.08 (s, 1); 9.02 (s, 1);8.46 (dd, 1); 8.38 (d, 1); 8.21-8.14 (m, 3); 7.97 (d, 1); 7.64 (dd, 1);7.23 (dd, 1); 4.23-4.14 (m, 1); 3.99-3.87 (m, 2); 3.22-3.19 (m, 1): 3.02(d, 1); 2.85 (dd, 1); 2.52 (s, 3); 2.30 (dd, 1); 2.11 (d, 1); 2.05-1.95(m, 1); 1.32 (s, 3); 1.21 (s, 3); 0.93 (d, 3); 0.86 (d, 3).

NH₄OAc standard conditions.

DAD R_(f)=1.67 min.

M+H=563.

Example 596,6-Dimethyl-4-{3-methyl-2-(S)-[(tetrahydro-furan-3-carbonyl)-amino]-butyl}-morpholine-3-(S)-carboxylicacid (6-chloro-9H-β-carbolin-8-yl)-amide

The desired compound was prepared following Method E from Intermediate61 and the appropriate acid.

¹H-NMR (300 MHz, methyl-d₃ alcohol-d): δ 0.87 (m, 6H), 1.25 (d, 3H),1.37 (d, 3H), 1.81 (m, 1H), 2.10-2.47 (m, 4H), 2.93 (m, 2H), 3.10 (m,1H), 3.26 (m, 1H), 3.80 (m, 1H), 3.86-4.07 (m, 6H), 7.86 (d, 1H), 8.10(m, 2H), 8.32 (d, 1H), 8.89 (s, 1H).

Retention Time (LC, method: ammonium acetate standard): 1.73 min. MS(M+H⁺): 542.

INTERMEDIATE 62:((S)-2-[(S)-5-(6-Chloro-4-methyl-9H-β-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-yl]-1-methyl-ethyl)-carbamicacid tert-butyl ester

A solution of(S)-4-((S)-2-tert-butoxycarbonylamino-propyl)-6,6-dimethyl-morpholine-3-carboxylicacid (3.316 g, 10.5 mmol) (prepared by reductively alkylating(S)-6,6-dimethyl-morpholine-3-carboxylic acid withN-(tert-butoxycarbonyl)-L-alanal) in anhydrous pyridine (75 mL) wasstirred at room temperature. 6-chloro-4-methyl-9H-β-carbolin-8-ylamine(1.869 g, 8.09 mmol) was added, followed by EDCI (2.894 g, 15.1 mmol).The reaction was stirred at room temperature for 14-18 hours underargon. The reaction was partially concentrated, diluted with H₂O (20 mL)and transferred to a reparatory funnel. The mixture was diluted withbrine (50 mL) and extracted with EtOAc (3×100 mL). The combined organiclayers were washed with brine, dried, filtered and concentrated toafford a dark residue. Column chromatography (0-8% MeOH/CH₂Cl₂) yieldedf(S)-2-[(S)-5-(6-Chloro-4-methyl-9H-β-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-yl]-1-methyl-ethyl)-carbamicacid tert-butyl ester as a light tan solid (2.688 g).

¹H-NMR (300 MHz, DMSO-d₆): δ 11.25 (s, 1) 9.94 (s, 1) 8.88 (s, 1) 8.18(s, 1) 8.02 (s, 1) 7.92 (s, 1) 6.73 (d, 1) 3.95-3.85 (m, 2) 3.66 (brs, 1) 3.16-3.08 (m, 1) 2.88 (d, 1) 2.76 (s, 3) 2.51-2.40 (m, 1) 2.23(dd, 1) 1.99 (d, 1) 1.34 (br s, 12) 1.17 (s, 3) 1.08 (d, 3).

NH₄OAc standard conditions.

ELSD R_(f)=2.07 min.

M+H=530.

Example 60(S)-6,6-Dimethyl-4-{(S)-2-[(2-methyl-pyridine-3-carbonyl)-amino]-propyl}-morpholine-3-carboxylicacid (6-chloro-4-methyl-9H-β-carbolin-8-yl)-amide

A solution of{(S)-2-[(S)-5-(6-Chloro-4-methyl-9H-β-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-yl]-1-methyl-ethyl}-carbamicacid tert-butyl ester (2.688 g, 5.08 mmol) in EtOH (60 mL) was stirredat room temperature. Concentrated HCl (10 mL) was added and the reactionstirred for 14 hours at room temperature under argon. The reaction wasconcentrated to afford a yellow solid (2.84 g). The solid was dissolvedin anhydrous pyridine (40 mL) and stirred at room temperature underargon. Triethylamine (2.20 mL, 15.7 mmol) and EDCI (1.39 g, 7.28 mmol)were added. The reaction mixture was stirred at room temperature for 10minutes and 2-methyl-nicotinic acid (0.868 g, 6.33 mmol) was added. Thereaction was stirred at room temperature for 14-18 hours and dilutedwith H₂O (40 mL). The mixture was poured into a separatory funnelcontaining H₂O (40 mL), brine (40 mL), and EtOAc (40 mL). The mixturewas shaken and the layers were separated. The aqueous layer wasextracted with EtOAc (2×40 mL) and the combined organic layers washedwith brine. The organic layer was dried, filtered, and concentrated. Theresulting residue was dissolved in EtOAc (10-20 mL) and added dropwiseto a stirring solution of 4:1 hexanes/Et₂O (300 mL). The precipitatewhich formed was collected via filtration and air dried to yield(S)-6,6-Dimethyl-4-{(S)-2-[(2-methyl-pyridine-3-carbonyl)-amino]-propyl}-morpholine-3-carboxylicacid (6-chloro-4-methyl-9H-β-carbolin-8-yl)-amide as a tan solid (3.151g).

¹H-NMR (300 MHz, DMSO-d₆): δ 11.28 (s, 1) 9.98 (s, 1) 8.81 (s, 1)8.45-8.39 (m, 1) 8.26 (d, 1) 8.13 (s, 1) 7.97 (s, 1) 7.89 (s, 1)7.64-7.56 (m, 1) 7.24-7.12 (m, 1) 4.25-4.10 (m, 1) 3.91-3.82 (m, 2)3.20-3.10 (m, 1) 2.94 (d, 1) 2.71 (s, 3) 2.61-2.49 (m, 1) 2.37 (s, 3)2.06-2.02 (m, 2) 1.30 (s, 3) 1.16 (d, 3) 1.15 (s, 3).

NH₄OAc standard conditions.

ELSD R_(f)=1.57 min. M+H=549.

INTERMEDIATE 63: 3,5-Difluoro-4-tributylstannanyl-pyridine

n-Butyl lithium (1.0 eq, 76 mmol, 47.6 mL, 1.6 M in hexanes) was addedvia dropping funnel to a solution of diisopropylamine (1.05 eq, 80 mmol,11.2 mL) in THF (300 mL) at −78° C. under nitrogen (N₂). The solutionwas stirred for 30 min at −78° C., then a solution of3,5-difluoropyridine (1.05 eq, 80 mmol, 9.2 g) in THF (20 mL) was addeddropwise via syringe. A beige precipitate was observed to form. Thereaction stirred at −78° C. for 90 min then tributyltin chloride (1.0eq, 76 mmol, 20.7 mL) was added dropwise via syringe and the resultingsolution allowed to warm to RT over 2 h. Water (5 mL) was added, thenroughly 250 mL of THF was removed on a rotary evaporator. The resultingmaterial was diluted with diethyl ether (350 mL) and washed successivelywith water (2×200 mL), saturated sodium chloride solution (1×150 mL),dried over magnesium sulfate, filtered and concentrated in vacuo toafford the 3,5-Difluoro-4-tributylstannanyl-pyridine as a colourless oil(27.5 g, 88%). This material was used crude without furtherpurification.

Retention Time (LC, method: ammonium acetate standard): 3.35 min. MS(M+H⁺): 406.

INTERMEDIATE 64: 4-Chloro-2-(3,5-difluoro-pyridin-4-yl)-phenylamine

Stille coupling: A dimethyl formamide (256 mL) solution of crudeIntermediate 63 (1.1 eq, 70 mmol, 27.5 g) and2-iodo-4-chloro-phenylamine (1.0 eq, 64 mmol, 16.2 g) was degassed withN₂ for 15 min. Dichlorobis(triphenylphosphine)palladium (II) (0.05 eq,3.2 mmol, 2.2 g) and copper (I) iodide (0.1 eq, 6.4 mmol, 1.2 g) wereadded and the suspension heated at reflux for 15 h under N₂. The mixturewas cooled to RT, filtered through a short plug of Celite® and thedimethyl formamide removed on a rotary evaporator. The crude materialwas dissolved in acetonitrile (300 mL), washed with hexanes (2×200 mL)then concentrated in vacuo. The material was then dissolved in ethylacetate (400 mL) and washed successively with water (2×200 mL),saturated sodium bicarbonate solution (1×200 mL), saturated sodiumchloride solution (200 mL), dried over magnesium sulfate, filtered andconcentrated in vacuo. The resulting solid was triturated with diethylether (50 mL) to remove the dark colour, then dissolved in the minimumvolume of methanol, filtered to remove an insoluble impurity andconcentrated in vacuo to afford the4-Chloro-2-(3,5-difluoro-pyridin-4-yl)-phenylamine as a tan solid (12.3g, ˜80%,) which was used in the subsequent step without furtherpurification.

¹H-NMR (300 MHz, dmso-d₆): δ 5.28 (s, 2H), 6.77 (d, 1H), 7.08 (d, 1H),7.19 (dd, 1H) and 8.58 (s, 2H).

Retention Time (LC, method: ammonium acetate standard): 1.70 min. MS(M+H⁺): not observed.

INTERMEDIATE 65: 6-Chloro-4-fluoro-9H-β-carboline

Sodium bis(trimethylsilyl)amide (3.0 eq, 130 mmol, 130 mL, 1.0M in THF)was added via dropping funnel to a solution of crude Intermediate 64(1.0 eq, 43 mmol, 10.4 g) in THF at RT under N₂. After stirring for 15 hthe excess base was quenched by the cautious addition of saturatedammonium chloride solution (100 mL) and the majority of the THF removedon a rotary evaporator. The resulting slurry was extracted with ethylacetate (400 mL then 2×200 mL), then the combined organics were washedsuccessively with saturated sodium bicarbonate solution (300 mL),saturated sodium chloride solution (300 mL), dried over sodium sulfateand filtered. Silica gel was added and the slurry concentrated on arotary evaporator. The material was purified using a Biotage Flash 75purification system (short column) eluting with 96:4dichloromethane/methanol to afford the 6-Chloro-4-fluoro-9H-β-carbolineas an off-white solid (7.8 g, 82%).

¹H-NMR (300 MHz, dmso-d₆): δ 7.71-7.61 (m, 3H), 8.11 (d, 1H) and 12.16(s, 1H).

Retention Time (LC, method: ammonium acetate standard): 1.70 min. MS(M+H⁺): 221.

INTERMEDIATE 66: 6-chloro-4-fluoro-9H-β-carbolin-8-ylamine

Sodium nitrate (1.5 eq, 53 mmol, 4.5 g) was added portionwise to asolution of Intermediate 65 (1.0 eq, 35 mmol, 7.8 g) in trifluoroaceticacid (200 mL) and the resulting mixture heated at 70° C. for 3 h. Aftercooling to RT the trifluoroacetic acid was removed on a rotaryevaporator to afford a crude solid which was suspended in a small volumeof methanol and added dropwise to a vigorously stirred mixture ofsaturated sodium bicarbonate solution (500 mL). The resulting slurry wasstirred for 15 min then the precipitated solids were collected bysuction filtration, washed with water (300 mL) and then dried in vacuoto afford 6-chloro-4-fluoro-8-nitro-9H-β-carboline (about 9.5 g) whichwas used in the subsequent step without further purification.

Retention Time (LC, method: ammonium acetate standard): 1.79 min. MS(M+H⁺): 266

Sulfated platinum (˜0.1 eq, 1 g) was added to a suspension of6-chloro-4-fluoro-8-nitro-9H-β-carboline (1.0 eq, 35 mmol, 9.3 g) andammonium formate (3.0 eq, 105 mmol, 6.6 g) in ethanol (175 mL) and theresulting mixture heated at 75° C. for 4 h. After cooling to RT themixture was filtered through a short plug of Celite® washing withcopious amounts of methanol, and then the filtrate concentrated in vacuoto afford a beige solid. The solid was suspended in the minimum volumeof methanol and added dropwise to a vigorously stirred mixture ofsaturated sodium bicarbonate solution and saturated sodium chloridesolution. After stirring for 15 min the precipitated solids werecollected by suction filtration, washed with water (200 mL) and dried invacuo to afford 6-chloro-4-fluoro-9H-β-carbolin-8-ylamine (5.8 g, 70% 2steps) as a beige powder.

¹H-NMR (300 MHz, dmso-d₆): δ 5.76 (s, 2H), 6.81 (d, 1H), 7.29 (d, 1H),8.24 (d, 1H), 8.82 (d, 1H), and 11.71 (s, 1H).

Retention Time (LC, method: ammonium acetate standard): 1.59 min. MS(M+H⁺): 236.

Example 61 4-(2-Acetylamino-propyl)-6,6-dimethyl-morpholine-3-carboxylicacid (6-chloro-4-fluoro-9H-β-carbolin-8-yl)-amide

The desired compound was prepared from Intermediate 66 and aceticanhydride.

¹H-NMR (300 MHz, dmso-d₆): δ 11.70 (s, 1H), 10.17 (s, 1H), 8.92 (s, 1H),8.34 (d, 1H), 7.95 (s, 2H), 7.81 (d, 1H), 4.05-3.95 (m, 1H), 3.92-3.83(m, 2H), 3.18-3.12 (m, 1H), 2.87 (d, 1H), 2.55-2.47 (m, 1H), 2.40-2.31(m, 1H), 2.03 (d, 1H), 1.78 (s, 3H), 1.32 (s, 3H), 1.63 (s, 3H) and 1.08(d, 3H).

Retention Time (LC, method: ammonium acetate standard): 1.73 min. MS(M+H⁺): 474.

Example 626,6-Dimethyl-4-{2-[(2-methyl-pyridine-3-carbonyl)-amino]-propyl}-morpholine-3-carboxylicacid (6-chloro-4-fluoro-9H-β-carbolin-8-yl)-amide

The desired compound was prepared according to Method E fromIntermediate 66 and methylnicotinic acid.

¹H-NMR (300 MHz, dmso-d₆): δ 11.63 (s, 1H), δ 10.10 (s, 1H), 5, 8.90 (s,1H), 6, 8.44 (d, 1H), 5, 8.34 (s, 1H), δ 8.29 (d, 1H), 7.94 (s, 2H),7.65 (d, 1H), 7.21 (dd, 1H), 4.25-4.15 (m, 1H), 3.97-3.88 (m, 2H),3.24-3.15 (m, 1H), 2.99 (d, 1H), 2.59 (t, 1H), 2.49 (s, 3H), 2.40 (dd,1H), 2.09 (d, 1H), 1.35 (s, 3H), 1.21 (d, 3H) and 1.20 (s, 3H).

Retention Time (LC, method: ammonium acetate standard): 1.67 min. MS(M+H⁺): 553.

Example 63[(S)-5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-yl]-aceticacid

To a suspension of (S)-6,6-Dimethyl-morpholine-3-carboxylic acid(6-chloro-9H-beta-carbolin-8-yl)-amide (2.6 g, 6.0 mmoles) in 60 ml ofmethanol was added 1.7 ml of triethylamine (2.0 eq.), sodiumcyanoborohydride (575 mg, 9.1 mmoles) and glyoxylic acid (780 mg, 8.5mmoles). The reaction mixture was stirred at ambient temperature for 1.5hrs. Water was added (5 ml) and the mixture was concentrated to a thickyellow slurry. More water was then added (30 ml) and the resultingslurry was stirred at ambient temperature for 10 min. and was filtered.The collected yellow solid was washed with water and dried under highvacuum to give 1.80 g (71%) of the desired product.

¹H-NMR (300 MHz, DMSO-d₅): δ 1.17 (s, 3H), 1.30 (s, 3H), 2.81 (d, 1H),3.34 (d, 1H), 3.46 (d, 1H), 3.56 (dd, 1H), 3.84-3.90 (m, 2H), 7.92 (s,1H), 8.15 (d, 1H), 8.21 (s, 1H), 8.36 (d, 1H), 9.01 (s, 1H), 10.28 (s,1H), 11.40 (s, 1H).

Retention Time (LC, method: ammonium acetate standard): 1.26 min.

MS (M+H⁺): 417.1.

METHOD F: Coupling procedure for reverse amides from[(8)-5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-yl]-aceticacid

[(S)-5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-yl]-aceticacid (1.0 mmol), EDCI (1.6 mmol) and the amine (1.2 mmol) to be coupledwere taken in a round-bottom flask and suspended in pyridine (5 ml). Theresulting mixture was stirred overnight. The pyridine was then removedunder reduced pressure and the residue was partitioned in EtOAc and 5%aqueous Na₂CO₃ solution. The separated aqueous phase was furtherextracted with EtOAc. The combined extracts were successively washedwith water and brine, dried over Na₂SO₄ and concentrated completely. Theresidue was purified on silica to give the desired product.

Example 64(S)-6,6-Dimethyl-4-(2-oxo-2-pyrrolidin-1-yl-ethyl)-morpholine-3-carboxylicacid (6-chloro-9H-beta-carbolin-8-yl)-amide

The desired compound was prepared according to Method F from[(S)-5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-yl]-aceticacid and pyrrolidine in 82% yield.

¹H-NMR (300 MHz, DMSO-d₆): δ 1.21 (s, 3H), 1.29 (s, 3H), 1.75-1.92 (m,4H), 2.46 (d, 1H), 2.77 (d, 1H), 3.35-3.68 (m, 7H), 3.94 (m, 2H), 8.08(s, 1H), 8.19 (d, 1H), 8.23 (s, 1H), 8.41 (d, 1H), 9.05 (s, 1H), 10.71(s, 1H), 11.51 (s, 1H).

Retention Time (LC, method: ammonium acetate standard): 1.75 min.

MS (M+H⁺): 470.3.

Example 65(S)-6,6-Dimethyl-4-(2-oxo-2-piperidin-1-yl-ethyl)morpholine-3-carboxylicacid (6-chloro-9H-beta-carbolin-8-yl)-amide

The desired compound was prepared according to Method F from[(S)-5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-yl]-aceticacid and piperidine in 90% yield.

¹H-NMR (300 MHz, DMSO-d₆): δ 1.17 (s, 3H), 1.29 (s, 3H), 1.35-1.60 (m,6H), 2.25 (d, 1H), 2.71 (d, 1H), 3.15 (d, 1H), 3.26 (dd, 1H), 3.37 (dd,1H), 3.45-3.65 (m, 2H), 3.65 (d, 1H), 3.70 (m, 1H), 3.89 (m, 2H) 7.95(d, 1H), 8.15 (d, 1H), 8.20 (d, 1H), 8.37 (d, 1H), 9.01 (s, 1H), 10.43(s, 1H), 11.32 (s, 1H)

Retention Time (LC, method: ammonium acetate standard): 1.86 min.

MS (M+H⁺): 484.3.

Example 66(S)-6,6-Dimethyl-4-(2-morpholin-4-yl-2-oxo-ethyl)-morpholine-3-carboxylicacid (6-chloro-9H-beta-carbolin-8-yl)-amide

The desired compound was prepared according to Method F from[(S)-5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-yl]-aceticacid and morpholine in 86% yield.

¹H-NMR (300 MHz, DMSO-d₆): δ 1.17 (s, 3H), 1.30 (s, 3H), 2.19 (d, 1H),2.71 (d, 1H), 3.03 (d, 1H), 3.16 (d, 1H), 3.22 (dd, 1H), 3.45-3.72 (m,7H), 3.80-3.98 (m, 3H), 7.94 (d, 1H), 8.15 (d, 1H), 8.21 (d, 1H), 8.37(d, 1H), 9.03 (s, 1H), 10.35 (s, 1H), 11.28 (s, 1H)

Retention Time (LC, method: ammonium acetate standard): 1.56 min.

MS (M+H⁺): 486.3.

Example 67(8)-4-{[(2-Hydroxy-ethyl)-methyl-carbamoyl]-methyl}-6,6-dimethyl-morpholine-3-carboxylicacid (6-chloro-9H-beta-carbolin-8-yl)-amide

The desired compound was prepared according to Method F from[(S)-5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-yl]-aceticacid and 2-methylamino-ethanol in 55% yield.

¹HNMR (300 MHz, DMSO-d₆): δ 1.21 (s, 3H), 1.30 (s, 3H), 2.39 (m, 1H),2.75 (bd, 1H), 2.96 (s, 1.5H), 3.17 (s, 1.5H), 3.50-3.65 (m, 2.5H),3.70-3.85 (m, 1.5H), 3.93 (m, 2H), 4.69 (m, 0.5H), 4.94 (m, 0.5H), 8.05(d, 1H), 8.18-8.25 (m, 2H), 8.42 (d, 1H), 9.06 (s, 1H), 10.68 (s, 1H),11.38 (s, 1H).

Retention Time (LC, method: ammonium acetate standard): 1.47 min.

MS (M+H⁺): 474.

Example 68(8)-6,6-Dimethyl-4-(pyridin-3-ylcarbamoylmethyl)-morpholine-3-carboxylicacid (6-chloro-9H-beta-carbolin-8-yl)-amide

The desired compound was prepared according to Method F from[(S)-5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-yl]-aceticacid and 3-aminopyridine. The product was isolated as hydrochloride saltin 55% yield.

¹HNMR (300 MHz, D₂O): δ 1.34 (s, 3H), 1.54 (s, 3H), 2.60 (d, 1H), 3.4(d, 1H), 3.50 (d, 1H), 3.70 (t, 1H), 3.81 (d, 1H) 4.23 (d, 2H), 7.69 (d,1H), 7.98 (d, 1H), 8.01 (d, 1H), 8.17 (d, 1H), 8.42 (d, 1H), 8.48-8.55(m, 3H), 9.09 (s, 1H), 9.30 (d, 1H).

Retention Time (LC, method: ammonium acetate standard): 1.56 min.

MS (M+H⁺): 493.2.

Example 69(S)-6,6-Dimethyl-4-{[(pyridin-4-ylmethyl)-carbamoyl]-methyl}-morpholine-3-carboxylicacid (6-chloro-9H-beta-carbolin-8-yl)-amide

The desired compound was prepared according to Method F from[(S)-5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-yl]-aceticacid and 4-(aminomethyl)pyridine. The product was isolated ashydrochloride salt in 53% yield.

¹HNMR (300 MHz, D₂O): δ 1.34 (s, 3H), 1.49 (s, 3H), 2.64 (d, 1H), 3.03(d, 1H), 3.48 (d, 1H), 3.72 (t, 1H), 3.74 (d, 1H), 4.15-4.25 (m, 2H),7.66 (d, 1H), 7.88 (s, 1H), 7.91 (s, 1H), 8.21 (d, 1H), 8.44 (d, 1H),8.53 (d, 1H), 8.56 (s, 1H), 8.58 (s, 1H), 9.08 (s, 1H).

Retention Time (LC, method: ammonium acetate standard): 1.47 min.

MS (M+H⁺): 507.3.

Example 704-[2-(4-Hydroxymethyl-piperidin-1-yl)-2-oxo-ethyl]-6,6-dimethyl-morpholine-3-carboxylicacid (6-chloro-9H-beta-carbolin-8-yl)-amide

[5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-yl]-aceticacid (200 mg, 0.48 mmol) and piperidin-4-yl-methanol (111 mg, 0.96 mmol)were dissolved in pyridine (4 mL). The resulting yellow solution wasstirred at room temperature for 10 min and then EDC (184 mg, 0.96 mmol)was added in a single portion. The reaction mixture was allowed to stirover night (16 h). Water (4 mL) was added and the mixture wasconcentrated under reduced pressure, The resulting residue waspartitioned between ethyl acetate (50 mL) and 1 M aqueous potassiumcarbonate. The aqueous layer was back-extracted with ethyl acetate (3×50mL) and the combined extracts were washed with water and brine, driedover sodium sulfate, filtered, and concentrated. The crude residue waspurified by silica gel chromatography (methylene chloride and methanolgradient) to afford pure4-[2-(4-hydroxymethyl-piperidin-1-yl)-2-oxo-ethyl]-6,6-dimethyl-morpholine-3-carboxylicacid (6-chloro-9H-beta-carbolin-8-yl)-amide as a yellow foam (152 mg,62%). The bis-HCl salt was prepared by adding 2 equivalents of conc. HClto an ethanolic solution of the freebase. Concentration, followed byether trituration afforded the salt as a free-flowing, yellow powder.

¹H-NMR (freebase, 300 MHz, CDCl₃) δ: 11.03 (d, 1H), 10.51 (d, 1H),8.97-8.80 (m, 1H), 8.47-8.27 (m, 2H), 7.94-7.76 (m, 2H), 4.84 (d, 1H),4.16-3.77 (m, 3H), 3.68-3.26 (m, 5H), 3.08 (ddd, 1H), 2.89-2.64 (m, 2H),2.50-2.37 (m, 2H), 1.98-1.70 (m, 3H), 1.38 (s, 3H), 1.25 (s, 3H),1.19-0.97 (m, 1).

MS (NH₄OAc standard conditions, ES+) e/z=514 (M+H)⁺

DAD R_(f)=1.51 min

Example 714-[2-(4-Hydroxy-piperidin-1-yl)-2-oxo-ethyl]-6,6-dimethyl-morpholine-3-carboxylicacid (6-chloro-9H-beta-carbolin-8-yl)-amide

The desired compound was synthesized using[5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-yl]-aceticacid and 4-hydroxypiperazine following Method F in 31% yield.

¹H-NMR (300 MHz, D₂O): δ 8.85 (1H, s), 8.19 (2H, s), 7.82 (1H, s), 7.44(1H, s), 4.15 (2H, m), 3.9-3.75 (4H, m), 3.6 (2H, m), 3.56 (2H, m), 3.12(1H, m), 2.9 (3H, m), 2.8 (1H, m), 1.7 (2H, m) 1.34 (3H, s), 1.19 (3H,s)

Retention time (LC, method: ammonium acetate standard): 1.5 min

MS (M+H⁺): 501

Example 72 4-Diethylcarbamoylmethyl-6,6-dimethyl-morpholine-3-carboxylicacid (6-chloro-9H-beta-carbolin-8-yl)-amide

The desired compound was made using of[5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-yl]-aceticacid and diethylamine following Method F in 60% yield.

¹H-NMR (300 MHz, D₂O): δ 8.87 (1H, s), 8.26 (2H, m), 7.8 (1H, s), 7.52(1H, s), 4.1 (3H, m), 3.8 (2H, m), 3.22 (2H, m), 3.0 (1H, d), 2.7 (1H,d), 1.3 (3H, s), 1.19 (3H, s), 0.09 (6H, m).

Retention time (LC, method: ammonium acetate standard): 1.96 min

MS (M+H⁺): 472

Example 736,6-dimethyl-4-[-2-(4-methyl-piperazin-1-yl)-2-oxo-ethyl]-morpholine-3-carboxylicacid (6-chloro-9H-beta-carbolin-8-yl)-amide

The desired compound was prepared using[5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-yl]-aceticacid and 1-methyl piperazine following Method F in 12% yield.

¹H-NMR (DMSO) δ 11.28 (1H, s), 10.35 (1H, s), 9.01 (1H, s), 8.37 (1H,s), 8.21-8.15 (2H, m), 7.9 (1H, s), 3.89 (2H, m), 3.7 (5H, m), 3.1 (2H,m), 2.85 (2H, m), 2.49 (3H, s), 2.4 (3H, m), 1.2 (3H, s), 1.16 (3H, s)

Retention time (LC, method: ammonium acetate standard): 1.34 min.

MS (M+H⁺): 500

Example 744-[2-(2,6-Dimethyl-morpholin-4-yl)-2-oxo-ethyl]-6,6-dimethyl-morpholine-3-carboxylicacid (6-chloro-9H-b-carbolin-8-yl)-amide

The desired compound was prepared using[5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-yl]-aceticacid and 2,6-dimethyl morpholine following Method F. Chromatographicpurification gave the desired product in 70-80% yield.

¹H-NMR (300 MHz, CDCl₃): δ 9.31 (s, 1H), 8.75 (d, 1H), 8.55 (d, 1H),8.37 (d, 1H), 8.14 (m, 1H), 4.73 (m, 2H), 4.96 (m, 1H), 4.37 (m, 2H),3.69 (m, 2H), 3.57 (m, 4H), 2.82 (t, 1H), 2.47 (t, 1H), 1.50 (d, 3H),1.42 (d, 3H), 1.18 (m, 6H)

Retention Time (LC, method: formic acid standard): 1.14 min (DiodeArray).

MS (M+H⁺): 514, (M−H⁺): 512

Example 751-{2-[5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-yl]-acetyl}-piperidine-4-carboxylicacid methyl ester

[5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-yl]-aceticacid (200 mg, 0.48 mmol) and methyl isonipecotate (137 mg, 130 uL, 0.96mmol) were dissolved in pyridine (4 mL) and stirred at room temperature10 min. EDC was added and the reaction mixture was allowed to stir atroom temperature over night (16 h). Additional methyl isonipecotate (137uL, 0.96 mmol) was added and the mixture was stirred an additional 24 h.Water (4 mL) was added and the mixture concentrated. The crude residuewas partitioned between ethyl acetate (75 mL) and 1 M aqueous potassiumcarbonate (50 mL). The aqueous phase was back-extracted with additionalethyl acetate (75+50 mL). The combined extracts were washed with waterand brine, dried over sodium sulfate, filtered, concentrated andpurified by silica gel chromatography (methylene chloride/methanolgradient) to afford the freebase as a yellow foam (150 mg, 57%). Thebis-HCl salt was prepared by adding 2 equivalents of conc. HCl to anethanolic solution of the freebase. Concentration, followed by ethertrituration afforded the salt as a free-flowing, yellow powder.

¹H-NMR (freebase, 300 MHz, CDCl₃) δ 11.04 (d, 1H), 10.48 (d, 1H), 8.95(s, 1H), 8.48-8.31 (m, 2H), 7.96-7.79 (m, 2H), 4.48 (dd, 1H), 4.13-3.92(m, 2H), 3.88-2.97 (m, 9H), 2.84-2.35 (m, 3H), 2.09-1.90 (m, 2H),1.85-1.52 (m, 2H), 1.38 (s, 3H), 1.23 (s, 3H).

MS (NH₄OAc standard conditions, ES+) e/z=, 542 (M+H)⁺

DAD R_(f)=1.72 min

Example 76(S)-4-[2-(3,3-Dimethyl-morpholin-4-yl)-2-oxo-ethyl]-6,6-dimethyl-morpholine-3-carboxylicacid (6-chloro-9H-beta-carbolin-8-yl)-amide-bis-hydrochloride salt

2,2-Dimethylmorpholine (3.0 g, 26.1 mmol) (prepared according to theprocedure of Cottle, D. L., et al. J. Org. Chem. 1946, 11, 286-291) wasdissolved in dichloromethane (60 mL). Triethylamine (3.6 mL, 2.66 g,26.1 mmol) was added and the reaction was cooled to −10° C. Bromoacetylchloride (2.2 mL, 4.08 g, 26.1 mmol) was added dropwise and the solutionwas warmed slowly to room temperature. The reaction was concentrated todryness in vacuo, redissolved in ethyl acetate and passed through a plugof silica gel. The eluant was concentrated to a yellow oil (3.45 g, 56%)that was carried to the next step.

Retention Time (LC, method: ammonium acetate standard): 1.20 min.

MS (M+H⁺): 237.

Intermediate 59 (60 mg, 0.14 mmol) was suspended in dichloromethane (3mL). A solution of 1M potassium carbonate (0.5 mL) was added. Theorganic layer was separated, dried over MgSO₄ and concentrated todryness. The free base was dissolved in DMF (1 mL) and stirred at roomtemperature. 2-Bromo-1-(3,3-dimethyl-morpholin-4-yl)-ethanone (30 mg,0.13 mmol) was dissolved in DMF (1 mL) and added dropwise. The reactionwas stirred for 3 hours at room temperature and concentrated to drynessin vacuo. Flash column chromatography (93:7 dichloromethane:methanol)afforded a yellow oil, which was dissolved in 4N HCl/dioxane (2 mL).Concentration in vacuo afforded the title compound as a yellow solid (22mg, 29%). ¹HNMR (300 MHz, MeOH-d₄): δ 1.35 (s, 3H)_(,) 1.41 (s, 3H),1.43 (s, 6H), 2.98 (m, 1H), 3.45 (m, 4H), 3.56 (m, 1H), 3.76 (m, 3H),4.30 (m, 4H), 8.04 (s, 1H), 8.39 (s, 1H), 8.52 (d, 1H), 8.73 (d, 1H),9.24 (s, 1H)

Retention Time (LC, method: ammonium acetate standard): 1.16 min.

MS (M+H⁺): 514

Example 774-[(Trans-4-hydroxy-cyclohexylcarbamoyl)-methyl]-6,6-dimethyl-morpholine-3-carboxylicacid (6-chloro-9H-beta-carbolin-8-yl)-amide

[5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-yl]-aceticacid (200 mg, 0.48 mmol), trans-4-aminocyclohexanol hydrochloride (145mg, 0.96 mmol) and diisopropylethylamine (124 mg, 167 uL, 0.96 mmol)were dissolved in pyridine (4 mL) and stirred at room temperature 10min. EDC (184 mg) was added and the reaction was stirred at roomtemperature over night (16 h). Water (2 mL) was added and the resultingmixture was concentrated under reduced pressure, The resulting residuewas diluted with 50 mL 1 M aqueous potassium carbonate and extractedwith ethyl acetate (75+2×50 mL). The combined extracts were washed withwater and brine, dried over sodium sulfate, filtered and concentrated.The crude residue was purified by silica gel chromatography(methanol/methylene chloride gradient) to afford pure4-[(trans-4-hydroxy-cyclohexylcarbamoyl)-methyl]-6,6-dimethyl-morpholine-3-carboxylicacid (6-chloro-9H-beta-carbolin-8-yl)-amide (148 mg, 59%) as a yellowfoam. The bis-HCl salt was prepared by adding 2 equivalents of conc. HClto an ethanolic solution of the freebase. Concentration, followed byether trituration afforded the salt as a free-flowing, yellow powder.

¹H-NMR (freebase, 300 MHz, CDCl₃) δ 11.71 (br s, 1H), 10.21 (s, 1H),9.06 (s, 1H), 8.45-8.27 (m, 2H), 8.08-7.84 (m, 2H), 6.36 (br s, 1H),4.13-3.81 (m, 3H), 3.72-3.56 (m, 1H), 3.51-3.37 (m, 2H), 3.03 (d, 1H),2.79-2.26 (m, 5H), 2.16-1.94 (m, 4H), 1.54-1.30 (m, 5H), 1.22 (s, 3H).

MS (NH₄OAc standard conditions, ES+) e/z=514 (M+H)⁺

DAD R_(f)=1.39 min

Example 78(S)-4-[2-((2R,5R)-2,5-Dimethyl-pyrrolidin-1-yl)-2-oxo-ethyl]-6,6-dimethyl-morpholine-3-carboxylicacid (6-chloro-9H-beta-carbolin-8-yl)-amide-bis-hydrochloride salt

(2R,5R)-2,5-Dimethyl-pyrrolidine hydrochloride (270 mg, 2.0 mmol)(prepared using the procedure of Masamune, S., et al. J. Org. Chem.1989, 54, 1756) was dissolved in dichloromethane (3 mL) and cooled to 0°C. Triethylamine (405 mg, 0.56 mL, 4.0 mmol) was added. Chloroacetylchloride (226 mg, 0.16 mL, 2.0 mmol) was dissolved in dichloromethane (1mL) and added dropwise. The mixture was warmed to room temperature andstirred an additional 30 minutes. The reaction was diluted withdichloromethane (5 mL), extracted with 1N HCl and brine, then dried overMgSO₄. The organic layer was concentrated to a brown oil. Wt.: 242 mg(82%).

Retention Time (LC, method: ammonium acetate standard): 1.24 min.

MS (M+H⁺): 176.5. The material was carried to the next step withoutfurther purification.

Intermediate 59 (495 mg, 1.15 mmol) was dissolved in a mixture ofacetonitrile (8 mL) and water (2 mL). Potassium carbonate (477 mg, 3.45mmol) was added and the reaction was warmed to 40° C.2-Chloro-1-((2R,5R)-2,5-dimethyl-pyrrolidin-1-yl)-ethanone (242 mg, 1.38mmol) (prepared as described above) was dissolved in acetonitrile (1 mL)and added dropwise. The reaction was warmed to 80° C. and stirredovernight. The reaction was cooled to 40° C. Sodium iodide (207 mg, 1.38mmol) was dissolved in acetone (1 mL) and added in one portion. Thereaction was stirred overnight at 40° C. The reaction was concentratedin vacuo and diluted with ethyl acetate (40 mL). The organic layer wasextracted twice with water, brine then dried over MgSO₄. The organiclayer was filtered, and concentrated to an orange foam in vacuo. Flashcolumn chromatography (95:5 dichloromethane:methanol) afforded a yellowsolid, which was dissolved in 4N HCl/dioxane (1 mL) and concentrated todryness. Trituration with ether afforded the title compound as a yellowsolid (18 mg, 3%).

¹H-NMR (300 MHz, MeOH-d₄): δ 1.23 (dd, 6H), 1.38 (s, 3H), 1.48 (s, 3H),1.65 (m, 2H), 2.23 (m, 2H), 3.18 (m, 1H), 3.50 (m, 1H), 3.66 (m, 1H),4.18 (m, 2H), 4.35 (m, 4H), 8.15 (s, 1H), 8.42 (s, 1H), 8.55 (d, 1H),8.78 (d, 1H), 9.29 (s, 1H).

Retention Time (LC, method: ammonium acetate standard): 2.02 min.

MS (M+H⁺): 498.

Example 794-{2-[4-(1-Hydroxy-1-methyl-ethyl)-piperidin-1-yl]-2-oxo-ethyl}-6,6-dimethyl-morpholine-3-carboxylicacid (6-chloro-9H-β-carbolin-8-yl)-amide

A solution of1-(2-[5-(6-chloro-9H-β-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-yl]-acetyl)-piperidine-4-carboxylicacid methyl ester (62 mg, 0.11 mmol) in a mixture of anhydrous ether andtoluene (1 ml: 1 ml) was stirred at 0° C. under N₂. To this solution wasslowly added methylmagnesium bromide (3.0 M in ether, 306 μL, 0.917mmol). The reaction mixture was stirred at room temperature overnight,and then quenched by adding saturated aqueous sodium bicarbonate. Theresulting mixture was further diluted with water (10 ml) and ethylacetate (30 ml). The aqueous layer was removed and extracted with ethylacetate (30×2 ml). The organic layers were combined, washed with brine,dried over magnesium sulfate, filtered and concentrated to afford ayellow solid (85 mg). The residue was purified by HPLC, to afford thepure product (13 mg, 20%).

¹H-NMR (300 MHz, HCDCl₃): δ 10.94 (d, 1H), 10.47 (d, 1H), 8.94 (s, 1H),8.41 (d, 1H), 8.32 (d, 1H), 7.89-7.81 (m, 2H), 4.91 (d, 1H), 4.04-3.91(m, 2H); 3.64-3.57 (m, 1H), 3.43 (s, 1H), 3.37-3.31 (m, 1H), 3.15-2.90(m, 1H), 2.77-2.58 (m, 2H), 2.47-2.41 (m, 1H), 2.00-1.86 (m, 2H),1.53-1.03 (m, 17H).

NH₄OAc standard conditions.

DAD R_(f)=1.85 min

M+H=542

Example 804-[2-(3,3-Dimethyl-4-oxo-piperidin-1-yl)-2-oxo-ethyl]-6,6-dimethyl-morpholine-3-carboxylicacid (6-chloro-9H-beta-carbolin-8-yl)-amide

4-Oxo-piperidine-1-carboxylic acid tert-butyl ester (5 g, 25 mmol) wasdissolved in tetrahydrofuran (100 mL) and the resulting solution wascooled to 0° C. Sodium hydride (60% in mineral oil, 2.10 g, 53 mmol) wasadded to the cooled solution in a single portion, and the resultingcloudy mixture was allowed to stir 10 min. Methyl iodide wassubsequently added and the mixture was allowed to warm to roomtemperature over several hours. Stirring continued over night (12 h).The light orange mixture was concentrated under reduced pressure. Theresidue was partitioned between ether and water. The aqueous phase wasback-extracted with additional ether. The combined extracts were washedwith water and brine, dried over sodium sulfate, filtered andconcentrated to a pale yellow solid. The solid was triturated with 4%ethyl acetate in hexanes (50 mL) to afford3,3-dimethyl-4-oxo-piperidine-1-carboxylic acid tert-butyl ester as acream-colored solid (1.8 g, 32%).

¹H-NMR (CDCl₃, 300 MHz) δ 3.73 (t, 2H), 3.43 (br s, 2H), 2.49 (t, 2H),1.49 (s, 9H), 1.13 (s, 6H).

The 3,3-dimethyl-4-oxo-piperidine-1-carboxylic acid tert-butyl esterthus prepared (450 mg, 1.97 mmol) was dissolved in methylene chloride(10 mL). Trifluoroacetic acid was added (305 uL) and the resultingsolution stirred at room temperature 2 h. Additional trifluoroaceticacid was added (300 uL) and the reaction stirred at room temperature 3days. The pale yellow solution was concentrated to afford an oilyresidue, which was triturated with ether. The solids were collected bysuction filtration and dried in vacuo. 3,3-Dimethyl-piperidin-4-one wasisolated and used as its trifluoroacetic acid salt (381 mg, 80%).

¹H-NMR (d₆-DMSO, 300 MHz) δ 3.44-3.33 (m, 4H), 2.63-2.57 (m, 2H), 1.11(s, 6H).

[5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-yl]-aceticacid (100 mg, 0.24 mmol), 3,3-dimethyl-piperidin-4-one trifluoroacetaticacid salt (116 mg, 0.48 mmol) and diisopropylethylamine (62 mg, 85 HL)were dissolved in pyridine (3 mL) and stirred 10 min. EDC (92 mg, 0.48mmol) was added and the mixture was stirred at room temperature 4 days.Water was added (3 mL) and the quenched reaction was concentrated. Theresidue was partitioned between ethyl acetate (50 mL) and 1 M aqueoussodium carbonate (50 mL). The aqueous phase was extracted withadditional ethyl acetate (50 mL), and the extracts were combined. Theextracts were then washed with water and brine, dried over sodiumsulfate, filtered, dried and concentrated under reduced pressure. Theresulting residue was purified by silica gel chromatography (chloroform,ethyl acetate, methanol gradient), affording4-[2-(3,3-dimethyl-4-oxo-piperidin-1-yl)-2-oxo-ethyl]-6,6-dimethyl-morpholine-3-carboxylicacid (6-chloro-9H-beta-carbolin-8-yl)-amide as a yellow foam (91 mg,73%). The bis-HCl salt was prepared by adding 2 equivalents of conc. HClto an ethanolic solution of the freebase.

Concentration, followed by ether trituration afforded the salt as afree-flowing, yellow powder.

¹H-NMR (CDCl₃, 300 MHz) δ 10.67 (s, 1H), 10.37 (d, 1H), 8.93 (s, 1H),8.41 (d, 1H), 8.15 (dd, 1H), 7.82 (d, 1H), 7.71 (d, 1H), 4.07-3.92 (m,3H), 3.83-3.89 (m, 6H), 2.79-2.68 (m, 1H), 2.62-2.40 (m, 3H), 1.41-1.36(m, 3H), 1.27-1.22 (m, 3H), 1.18-1.13 (m, 3H), 1.07-0.99 (m, 3H).

MS (NH₄OAc standard conditions, ES+) e/z=526 (M+H)⁺

DAD R_(f)=1.79 min

Example 816,6-Dimethyl-4-(2-oxo-2-pyrrolidin-1-yl-ethyl)-morpholine-3-carboxylicacid (6-chloro-4-methyl-9H-β-carbolin-8-yl)-amide

The desired compound was prepared according to Methods C, E and F from6-chloro-4-methyl-9H-β-carbolin-8-ylamine (Intermediate 54) andpyrrolidine.

¹H-NMR (300 MHz, MeOD-d₄) δ 9.17 (s, 1) 8.39 (s, 1) 8.26 (d, 1) 8.17(d, 1) 4.74-4.52 (m, 3) 4.47-4.30 (m, 1) 3.80-3.52 (m, 3) 3.48-3.34 (m,4) 3.01 (s, 3) 1.76-1.55 (m, 4) 1.50 (s, 3) 1.43 (s, 3).

NH₄OAc standard conditions ELSD R_(f)=2.01 min.

M+H=498

Biological Testing

Compounds of this invention are effective inhibitors of IκB kinase(IKK), and therefore, are useful for treating conditions caused oraggravated by the activity of this kinase. The in vitro and in vivo IκBkinase inhibitory activities of the compounds of formula I may bedetermined by various procedures known in the art. The potent affinitiesfor IκB kinase exhibited by the inventive compounds can be measured asan IC₅₀ value (in nM), which is the concentration (in nM) of compoundrequired to provide 50% inhibition of IκB kinase.

Following are examples of assays that can be useful for evaluating andselecting a compound that modulates IKK.

Assay for Measuring IκB Kinase Enzyme Inhibition

An in vitro assay for detecting and measuring inhibition activityagainst IκB kinase complex by candidate pharmacological agents canemploy a polypeptide spanning both Ser³² and Ser³⁶ of IκB (SwissProtAccession No. P25963, Swiss Institute of Bioinformatics, Geneva,Switzerland) and an agent for detection of the phosphorylated product,e.g. a specific antibody binding only to the phosphorylated form of thepolypeptide, being either monoclonal or polyclonal (e.g.,commercially-available anti-phospho-serine³² IκB antibodies). In theexample of detecting the phosphorylated product by ananti-phosphoserine³² IκB antibody, once the antibody-phospho-polypeptidecomplex is formed, the complex can be detected by a variety ofanalytical methods (e.g., radioactivity, luminescence, fluorescence, oroptical absorbance). For the use of the DELFIA (Dissociation EnhancementLanthanide Fluorescence Immunoassay) method (time-resolved fluorometry,Perkin Elmer Life and Analytical Sciences Inc., Boston, Mass.), thecomplex can be immobilized either onto a biotin-binding plate (e.g.,Neutravidin coated plate) and detected with a secondary antibodyconjugated to Europium, or onto an antibody-binding plate (e.g.,Protein-A coated plate) and detected with biotin-binding proteinconjugated to Europium (e.g., Streptavidin-Europium). The level ofactivity can be correlated with a standard curve using syntheticphosphopeptides corresponding to the substrate polypeptide. How toprepare materials for and conduct this assay are described in moredetail below.

Isolation of the IκB Kinase Complex

An IκB-α kinase complex was prepared by first diluting 10 ml of HeLa S3cell-extracts 5100 fraction (Lee et al. (1997) Cell 88:213-222) with 40ml of 50 mM HEPES pH 7.5. Then, 40% ammonium sulfate was added andincubated on ice for 30 minutes. The resulting precipitated pellet wasredissolved with 5 ml of SEC buffer (50 mM HEPES pH 7.5, 1 mM DTT, 0.5mM EDTA, 10 mM 2-glycerophosphate), clarified by centrifugation at20,000×g for 15 min., and filtrated through a 0.22 μm filter unit. Thesample was loaded onto a 320 ml SUPEROSE-6 gel filtration FPLC column(Amersham Biosciences AB, Uppsala, Sweden) equilibrated with a SECbuffer operated at 2 ml/min flow rate at 4° C. Fractions spanning the670-kDa molecular-weight marker were pooled for activation. Akinase-containing pool was then activated by incubation with 100 nMMEKK1Δ (Lee et al. (1997) Cell 88:213-222), 250 μM MgATP, 10 mM MgCl₂, 5mM DTT, 10 mM 2-glycerophosphate, 2.5 μM Microcystin-LR, for 45 minutesat 37° C. The activated enzyme was stored at −80° C. until further use.

Measurement of IκB Kinase Phosphotransferase Activity

At the per well of a 96 well plate, compounds of various concentrationsin 5 μL of 20% DMSO were preincubated for 30 minutes at 25° C. with 40μL of activated enzyme diluted 1:25 with assay buffer (50 mM Hepes pH7.5, 5 mM DTT, 10 mM MgCl₂, 10 mM 2-glycerophosphate, 2 μMMicrocystin-LR, 0.1% Bovine Serum Albumin). 5 μL of peptide substrate(biotin-(CH₂)-6-DRHDSGLD(phosphoS)MKD-CONH₂) at 200 μM+500 μM ATP wereadded to each well and incubated for 1 hour before quenching with 50 μLof 50 mM Hepes pH 7.5, 0.1% BSA, 100 mM EDTA. 5 μL of quenched kinasereaction were transferred to a Protein A plate (Pierce Biotechnology,Inc., Rockford, Ill., USA) containing 90 μL of anti-phospho IκB S32/S36antibody (Cell Signaling Technologies Beverly, Mass., USA) at 2 μg/ml.Samples were incubated for 2 hours with shaking. Following 3 washes withPBS+0.05% Tween20, 90 μL of streptavidin linked europium chelate (PerkinElmer Life and Analytical Sciences, Boston, Mass., USA) at 0.1 μg/mlwere added to each well and incubated for 1 hour with shaking. Following3 washes with PBS 0.05% Tween20, 100 μL of DELFIA Enhancement Solution(Perkin Elmer Life and Analytical Sciences, Boston, Mass., USA) wereadded to each well. An europium signal was read with an excitation of330 nM and emission of 615 nM on a Wallac Victor plate reader (PerkinElmer Life and Analytical Sciences, Boston, Mass.). As the assay waspreviously shown to be linear with respect to enzyme concentration andtime for the enzyme dilution tested, levels of europium signal were usedto determine the inhibition activity of candidate pharmacologicalagents.

The compounds of the invention were active inhibitors of the IKKcomplex. It will be appreciated that compounds of this invention canexhibit IκB kinase inhibitor activities of varying degrees. Followingassay procedures such as the in vitro and cell-based assays describedherein, the IκB kinase inhibition average IC₅₀ values for the inventivecompounds were generally below about 10 micromolar, preferably, belowabout 1.0 micromolar and more preferably below about 100 nanomolar. Theinventive compounds were also selective for inhibiting IKK-2 as opposedto IKK-1.

Cellular Assays Multiple Myeloma (MM) Cell Lines and Patient-Derived MMCells Isolation

RPMI 8226 and U266 human MM cells were obtained from American TypeCulture Collection (Manassas, Va.). All MM cell lines were cultured inRPMI-1640 containing 10% fetal bovine serum (FBS, Sigma-Aldrich Co., St.Louis, Mo.), 2 mM L-glutamine, 100 U/mL penicillin (Pen) and 100 μg/mLstreptomycin (Strep) (GIBCO brand cell culture products available fromInvitrogen Life Technologies, Carlsbad, Calif.). Patient-derived MMcells were purified from patient bone marrow (BM) aspirates usingROSETTESEP (B cell enrichment kit) separation system (StemCellTechnologies, Vancouver, Canada). The purity of MM cells was confirmedby flow cytometry using PE-conjugated anti-CD138 antibody (BDBiosciences, Bedford, Mass.).

Bone Marrow Stroma Cell Cultures

Bone marrow (BM) specimens were obtained from patients with MM.Mononuclear cells (MNCs) separated by Ficoll-Hipaque densitysedimentation were used to established long-term BM cultures aspreviously described (Uchiyama et al., Blood 1993, 82:3712-3720). Cellswere harvested in Hank's Buffered Saline Solution (HESS) containing0.25% trypsin and 0.02% EDTA, washed, and collected by centrifugation.

Cell Proliferation Via Measurement of DNA-Synthesis Rate

Proliferation was measured as previously described (Hideshima et al.,Blood 96:2943 (2000)). MM cells (3×10⁴ cells/well) were incubated in96-well culture plates (Corning Life Sciences, Corning, N.Y.) in thepresence of media or an IKK inhibitor of this invention for 48 h at 37°C. DNA synthesis was measured by [³H]-thymidine ([³H]-TdR, New EnglandNuclear division of Perkin Elmer Life and Analytical Sciences, Boston,Mass.) incorporation into dividing cells. Cells were pulsed with [³H]TdR(0.5 μCi/well) during the last 8 h of 48 h cultures. All experimentswere performed in triplicate.

MTT Cell Viability Assay

The inhibitory effect of the present compounds on MM growth was assessedby measuring the reduction of yellow tetrazolium MTT(3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) bymetabolically active cells (J. Immunol. Methods 174: 311-320, 1994).Cells from 48 h cultures were pulsed with 10 μL of 5 mg/mL MTT to eachwell for the last 4 h of the 48 h cultures, followed by 100 μLisopropanol containing 0.04N HCl. Absorbance was measured at 570 nmusing a spectrophotometer (Molecular Devices Corp., Sunnyvale Calif.).

NF-κB Activation Via Electrophoretic Mobility Shift Assay

Electrophoretic mobility shift analyses (BMSA) were carried out aspreviously described (Hideshima et al., Oncogene 2001, 20:4519).Briefly, MM cells were pre-incubated with an IKK inhibitor of thisinvention (10 μM for 90 min) before stimulation with TNF-α (5 ng/mL) for10 to 20 min. Cells were then pelleted, resuspended in 400 μL ofhypotonic lysis buffer (20 mM HEPES, pH 7.9, 10 mM KCl, 1 mM EDTA, 0.2%Triton X-100, 1 mM Na₃VO₄, 5 mM NaF, 1 mM PMSF, 5 μg/mL leupeptin, 5μg/mL aprotinin), and kept on ice for 20 min. After centrifugation(14000 g for 5 min) at 4° C., the nuclear pellet was extracted with 100μL hypertonic lysis buffer (20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA,1 mM Na₃VO₄, 5 mM NaF, 1 mM PMSF, 5 μg/mL leupeptin, 5 μg/mL aprotinin)on ice for 20 min. After centrifugation (14000 g for 5 min) at 4° C.,the supernatant was collected as nuclear extract. Double-stranded NF-κBconsensus oligonucleotide probe (5′-GGGGACTTTCCC-3′, Santa CruzBiotechnology Inc., Santa Cruz Calif.) was end-labeled with [(³²P]ATP(50 μCi at 222 TBq/mM; New England Nuclear division of Perkin Elmer Lifeand Analytical Sciences, Boston, Mass.). Binding reactions containing 1ng of oligonucleotide and 5 μg of nuclear protein were conducted at roomtemperature for 20 min in a total volume of 10 μL of binding buffer (10mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM MgCl₂, 0.5 mM EDTA, 0.5 mM DTT, 4%glycerol (v/v), and 0.5 μg poly(dI-dC) (Amersham Biosciences AB,Uppsala, Sweden). For supershift analysis, 1 μg of anti-p65 NF-κB Ab wasadded 5 min before the reaction mixtures, immediately after addition ofradiolabeled probe. The samples were loaded onto a 4% polyacrylamidegel, transferred to Whatman paper (Whatman International, Maidstone,U.K.), and visualized by autoradiography.

Diffuse Large B-Cell Lymphoma (DLBCL) Cell Proliferation Assay

ABC-like (LY3 and Ly10) and GCB-like (Ly7 and Ly19) DLBCL cell lines(Alizadeh et al (2000) Nature 403:503-511; Davis et al. (2001) J. Exp.Med. 194:1861-1874) were maintained in growth medium (GM, Iscove'sDMEM+10% FBS) by passaging cells twice per week. Cells were starvedovernight in Iscove's DMEM medium+0.5% FBS overnight before plated inproliferation assay. On the day of the assay, cells were counted andviability was checked using Trypan Blue staining. For the Ly3 and Ly10cells, 5000 cell were plated in GM per well in a 96-well plate. The Ly7and Ly19 cells were plated at 10,000 cells per well. IKK inhibitors werefirst dissolved in DMSO and then diluted in GM to reach the finalconcentrations of 80 μM-0.01 μM. Each concentration was plated intriplicate. Cell viability was determined using a standard WST-1 cellviability assay (Roche Applied Science, Indianapolis, Ind.).

Human Peripheral Blood Monocyte (PBMC) Cytokine Release Assay

Human PBMC was purified from normal donor whole blood by Ficoll gradientmethod. After a PBS wash, PBMC were re-suspended in AIM-V medium.Serially diluted IKK inhibitors of this invention in 100% DMSO was addedat 1 μl to the bottom of a 96-well plate and mixed with 180 μl 4.5×10⁵PBMC in AIM-V media per well. After preincubating PBMC with inhibitor at37° C. for 40 min, cells were stimulated with 20 μl of either with LPS(100 ng/ml) or with anti-CD3 (0.25 μg/ml) and anti-CD28 (0.25 μg/ml)(Pharmingen division of BD Biosciences, Bedford, Mass.) at 37° C. for 5hours. The supernatants were collected and assessed for IL-1β or TNF-αrelease using standard commercially available ELISA kits.

Human Chondrocyte Matrix Metalloproteases (MMPs) Release Assay

Human chondrocyte cell line SW1353 (ATCC, Manassas, Va.) was culturedcontaining 10% fetal bovine serum (Hyclone, Logan, Utah), 2 mML-glutamine (GIBCO brand cell culture products available from InvitrogenLife Technologies, Carlsbad, Calif.) and 1% Pen/Strep (GIBCO). Cellswere seeded in 96-well Poly-D-Lysine plate (BD BIOCOAT, Black/Clearbottom, BD Biosciences, Bedford, Mass.). Serially diluted IKK inhibitorsat 1 μl were added to each well of 96-well plates and mixed with 180 μl4.5×10⁵ chondrocytes per well. After pre-incubating cells with compoundsfor 1 hr at 37° C., cells were stimulated with 20 μl IL-1β (10 ng/mL,R&D Systems Inc.) at 37° C. for 24 hrs. The supernatants were thencollected and assessed for production of matrix metalloproteinases(MMPs) using commercially available ELISA kits.

Human Fibroblast Like Synoviocyte (HFLS) Assay

HFLS isolated from RA synovial tissues obtained at joint replacementsurgery was provided by Cell Applications Inc. (San Diego, Calif.). IKKinhibitors of the invention were tested for their ability to block theTNF- or IL-1β-induced release of IL-6 or IL-8 from these cells usingcommercially available ELISA kits. Cell culture conditions and assaymethods were described in Aupperle et al., Journal of Immunology,163:427-433 (1999).

Human Cord Blood Derived Mast Cell Assay

Human cord blood was obtained from Cambrex (Walkersville, Md.). Mastcells were differentiated and cultured in a manner similar to thatdescribed by Hsieh et al., J. Exp. Med., 193:123-133 (2001). IKKinhibitors of the invention were tested for their ability to block theIgE- or LPS-induced TNFα release using commercially available ELISAkits.

Osteoclast Differentiation and Functional Assays

Human osteoclast precursors were obtained as cryopreserved form fromCambrex (Walkersville, Md.). The cells were differentiated in culturebased on instructions from the manufacturer. IKK inhibitors of theinvention were tested for their ability to block the differentiation,bone resorption and collagen degradation as described previously (seeKhapli, S. M., Journal of Immunol, 171:142-151 (2003); Karsdal, M. A., JBiol Chem, 278:44975-44987 (2003); and Takami, M., Journal of Immunol,169:1516-1523 (2002)).

Rat Models for Rheumatoid Arthritis

Certain compounds of this invention were found to be active in one ormore rat models for rheumatoid arthritis. Such testing is known in theliterature and include a standard rat LPS model as described in Conwayet al., “Inhibition of Tumor Necrosis Factor-α (TNF-α) Production andArthritis in the Rat by GW3333, a Dual Inhibitor of TNF-ConvertingEnzyme and Matrix Metalloproteinases”, J. Pharmacol. Exp. Ther. 298(3),900-908 (2001); a rat adjuvant induced arthritis model as described inPharmacological Methods in the Control of Inflammation (1989) p 363-380“Rat Adjuvant Arthritis: A Model of Chronic Inflammation” Barry M.Weichman author of book chapter {Alan R. Liss Inc Publisher}; and a ratcollagen induced arthritis model as described in Pharmacological Methodsin the Control of Inflammation (1989) p 395-413 “Type II CollagenInduced Arthritis in the Rat” DE Trentham and RA Dynesuis-Trenthamauthors of book chapter {Alan R. Liss Inc Publisher}, See also, “AnimalModels of Arthritis: Relevance to Human Disease” (1999) by A. Bendele,J. McComb, T. Gould, T. McAbee, G. Sennello, E. Chlipala and M. Guy.Toxicologic Pathology Vol 27 (1) 134-142.

Based on the results of one or more rat models such as the onesdescribed above, compounds of formula II-C were found to be surprisinglysuperior compared to other compounds where Ring A is a pyridine ring.Also in the rat models, compounds of formula III-A-a, especiallycompounds of formula III-A-aa, were found to be surprisingly superiorcompared to other compounds where Ring A is a morpholine ring.

While we have described a number of embodiments of this invention, it isapparent that our basic examples may be altered to provide otherembodiments, which utilize the compounds and methods of this invention.Therefore, it will be appreciated that the scope of this invention is tobe defined by the appended claims rather than by the specificembodiments, which have been represented by way of example.

1. A compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein: Ring A isselected from the group consisting of: (a) a pyridinyl or pyrimidinylring that is substituted by (i) —CH₂C(O)-G and 0-1 R^(6a) or (ii) 1-2R^(6a), and (b) a morpholinyl, piperidinyl, piperazinyl, pyrrolidinyl,pyranyl, tetrahydrofuranyl, cyclohexyl, cyclopentyl or thiomorpholinylring that is substituted by (i) —C(R⁹)₃, —W-G, or -G, (ii) 0-4 R^(6b)and (iii) 0-1 oxo groups on a ring carbon or 0-2 oxo groups on a ringsulfur; each R^(6a) is independently selected from C₁₋₆ aliphatic, halo,alkoxy, or amino; each R^(6b) is independently selected from C₁₋₃aliphatic or —N(R⁷)₂, and two R^(6b) on the same or an adjacent carbonoptionally are taken together with the intervening carbon(s) to form a5-6 membered ring having 1-2 ring heteroatoms selected from N, O or S; Wis -Q-, -Q-C(O)—, —C(R⁹)₂—C(R⁹)(R¹²)—, or —C(R⁹)₂-[C(R⁹)(R¹²)]₂—; Q is—C(R⁹)₂— or —C(R⁹)₂C(R⁹)₂—; G is —OH, —NR⁴R⁵, —N(R⁹)CONR⁴R⁵, —N(R⁹)SO₂(C₁₋₃ aliphatic), —N(R⁹)COCF₃—N(R⁹)CO(C₁₋₆ aliphatic),—N(R⁹)CO(heterocyclyl), —N(R⁹)CO(heteroaryl)) —N(R⁹)CO(aryl), a 3-7membered heterocyclyl ring, or a 5-6 membered heteroaryl, wherein eachof the heteroaryl, aryl and heterocyclyl moieties of G is optionallysubstituted by 1-3 R¹⁰; R¹ is hydrogen, halo, C₁₋₃ aliphatic, amino,cyano, (C₁₋₃ alkyl)₁₋₂ amino, C₁₋₃ alkoxy, —CONH₂, —NHCOCF₃, or —CH₂NH₂;R² is hydrogen, halo, C₁₋₃ aliphatic, —CF₃; R³ is hydrogen, halo, C₁₋₆aliphatic, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, hydroxy, amino, cyano, or (C₁₋₆alkyl)₁₋₂ amino; R⁴ is hydrogen, 3-7 membered heterocyclyl, or C₁₋₆aliphatic; R⁵ is hydrogen, C₁₋₆ aliphatic group or a 3-7 memberedheterocyclic ring having 1-2 ring heteroatoms selected from N, O, or S,wherein R⁵ is optionally substituted by halo, —OR′, —CN, —SR^(O),—S(O)₂R⁸, —S(O)₂N(R⁷)₂, —C(O)R⁷, —CO₂R⁷, —N(R⁷)₂, —C(O)N(R⁷)₂,—N(R⁷)C(O)R⁷, —N(R⁷)CO₂R¹³, or —N(R⁷)C(O)N(R⁷)₂; each R⁷ isindependently selected from hydrogen or C₁₋₄ aliphatic, or two R⁷ on thesame nitrogen atom are taken together with the nitrogen to form a 5-6membered heteroaryl or heterocyclyl ring; each R⁸ is independentlyselected from C₁₋₄ aliphatic; each R⁹ is independently selected fromhydrogen or C₁₋₃ aliphatic; each R¹⁰ is independently selected from oxo,—R¹¹, -T-R¹¹, or -V-T-R¹¹; each R¹¹ is independently selected from C₁₋₆aliphatic, halo, —S(O)₂N(R⁷)₂, —OR⁷, —CN, —SR⁸, —S(O)₂R⁸, —C(O)R⁷,—CO₂R⁷, —N(R⁷)₂, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)CO₂R⁷, or—N(R⁷)C(O)N(R⁷)₂; T is a straight or branched C₁₋₄ alkylene chain; V is—O—, —N(R⁷)—, —S—, —S(O)—, —S(O)₂—, —C(O)—, or —CO₂—, and R¹² ishydrogen or an amino acid side chain.
 2. The compound of claim 1 whereRing A is a 3-pyridinyl or 5-pyrimidinyl ring substituted by 1-2 R^(6a)groups.
 3. The compound of claim 2 having formula II-C:

wherein: Y is N or CH; R¹ is hydrogen, halo, C₁₋₃ aliphatic, amino,cyano, (C₁₋₃ alkyl)₁₋₂ amino, C₁₋₃ alkoxy, —CONH₂, —NHCOCF₃, or —CH₂NH₂;R² is hydrogen, halo, C₁₋₃ aliphatic, —CF₃; R³ is hydrogen, halo, C₁₋₆aliphatic, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, hydroxy, amino, cyano, or (C₁₋₆alkyl)₁₋₂ amino; and R^(6a) is selected from C₁₋₆ aliphatic or halo. 4.The compound of claim 3 where R^(6a) is methyl.
 5. The compound of claim4 where R¹ is hydrogen, methyl, amino or fluoro; R² is hydrogen or halo;and R³ is hydrogen, halo or C₁₋₄alkoxy.
 6. The compound of claim 5 whereY is CH.
 7. The compound of claim 5 where Y is N.
 8. The compound ofclaim 1 where Ring A is selected from morpholinyl, piperidinyl,piperazinyl, pyrrolidinyl, pyranyl, tetrahydrofuranyl, cyclohexyl,cyclopentyl or thiomorpholinyl and where Ring A is substituted by (i)—C(R⁹)₃ or —W-G, (ii) 0-4 R^(6b) and (iii) 0-1 oxo groups on a ringcarbon or 0-2 oxo groups on a ring sulfur.
 9. The compound of claim 8where the —W-G or —C(R⁹)₃ substituent on Ring A is ortho to the positionwhere the beta-carboline portion is attached.
 10. A compound of formulaIII-A:

or a pharmaceutically acceptable salt thereof, wherein: Ring A issubstituted by 0-4 R^(6b); each R^(6b) is independently selected fromC₁₋₃ aliphatic or —N(R⁷)₂, and two R^(6b) on the same or an adjacentcarbon optionally are taken together with the intervening carbon(s) toform a 5-6 membered ring having 1-2 ring heteroatoms selected from N, Oor S; W is -Q-, -Q-C(O)—, —C(R⁹)₂—C(R⁹)(R¹²)—, or—C(R⁹)₂—[C(R⁹)(R¹²)]₂—; Q is —C(R⁹)₂— or —C(R⁹)₂C(R⁹)₂—; G is —OH,—NR⁴R⁵, —N(R⁹)CONR⁴R⁵, —N(R⁹)SO₂ (C₁₋₃ aliphatic), —N(R⁹)COCF₃,—N(R⁹)CO(C₁₋₆ aliphatic), —N(R⁹)CO(heterocyclyl)-N(R⁹)CO(heteroaryl)—N(R⁹)CO(aryl), a 3-7 membered heterocyclyl ring, or a 5-6 memberedheteroaryl, wherein each of the heteroaryl, aryl and heterocyclylmoieties of G is optionally substituted by 1-3 R¹⁰; R¹ is hydrogen,halo, C₁₋₃ aliphatic, amino, cyano, (C₁₋₃ alkyl)₁₋₂ amino, C₁₋₃ alkoxy,—CONH₂, —NHCOCF₃, or —CH₂NH₂; R² is hydrogen, halo, C₁₋₃ aliphatic,—CF₃; R³ is hydrogen, halo, C₁₋₆ aliphatic, C₁₋₆ haloalkyl, C₁₋₆ alkoxy,hydroxy, amino, cyano, or (C₁₋₆ alkyl)₁₋₂ amino; R⁴ is hydrogen, 5-6membered heterocyclyl, or C₁₋₆ aliphatic; R⁵ is hydrogen, C₁₋₆ aliphaticgroup or a 5-6 membered heterocyclic ring having 1-2 ring heteroatomsselected from N, O, or S, wherein R⁵ is optionally substituted by halo,—OR⁷, —CN, —SR⁸, —S(O)₂R⁸, —S(O)₂N(R⁷)₂ (O)R⁷, —CO₂R⁷, —N(R⁷)₂,—C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)CO₂R⁸, or —N(R⁷)C(O)N(R⁷)₂; each R⁷ isindependently selected from hydrogen or C₁₋₄ aliphatic, or two R⁷ on thesame nitrogen atom are taken together with the nitrogen to form a 5-6membered heteroaryl or heterocyclyl ring; each R⁸ is independentlyselected from C₁₋₄ aliphatic; each R⁹ is independently selected fromhydrogen or C₁₋₃ aliphatic; each R¹⁰ is independently selected from oxo,—R¹¹, -T-R¹¹, or -V-T-R¹¹; each R¹¹ is independently selected from C₁₋₆aliphatic, halo, —S(O)₂N(R⁷)₂, —CN, —SR⁸, —S(O)₂R⁸, —C(O)R⁷, —CO₂R⁷,—N(R⁷)₂, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)CO₂R⁷, or —N(R⁷)C(O)N(R⁷)₂; Tis a straight or branched C₁₋₄ alkylene chain; V is —O—, —N(R⁷)—, —S—,—S(O)—, —S(O)₂—, —C(O)—, or —CO₂—; and R¹² is hydrogen or an amino acidside chain.
 11. The compound of claim 10 having the formula (S)-III-A:

where n is 0-4 and R¹, R², R³, W, G and R^(6b) are as defined in claim10.
 12. The compound of claim 11 where: R¹ is hydrogen, halo, methyl oramino; R² is hydrogen, methyl or halo; R³ is hydrogen, halo, alkoxy, or(C₁₋₆ aliphatic)₂ amino; n is 0-2; R^(6b) is C₁₋₃ aliphatic; W is -Q-,-Q-C(O)—, —C(R⁹)₂—C(R⁹)(R¹²)—, or —C(R⁹)₂—[C(R⁹)(R¹²)]₂—; Q is —C(R⁹)₂—or —C(R⁹)₂C(R⁹)₂—; G is —NR⁴R⁵, —N(R⁹)C(O)NR⁴R⁵, —N(R⁹)SO₂(C₁₋₃aliphatic), —N(R⁹)C(O)CF₃, —N(R⁹)CO(C₁₋₆ aliphatic), and—N(R⁹)CO(heterocyclyl)-N(R⁹)CO(heteroaryl) —N(R⁹)CO(aryl), a 5-6membered heterocyclyl ring, or a 5-6 membered heteroaryl, wherein eachof the heteroaryl, aryl and heterocyclyl moieties of G is optionallysubstituted by 1-3 R¹⁰; R⁴ is hydrogen or C₁₋₆ aliphatic; R⁵ is hydrogenor a C₁₋₆ aliphatic group that is optionally substituted by halo, —OR⁷,—CN, —SR⁸, —S(O)₂R⁸, —S(O)₂N(R⁷)₂, —C(O)R⁷, —CO₂R⁷, —N(R⁷)₂,—C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)CO₂R⁸, or —N(R⁷)C(O)N(R⁷)₂; each R⁷ isindependently selected from hydrogen or C₁₋₄ aliphatic, or two R⁷ on thesame nitrogen atom are taken together with the nitrogen to form a 5-6membered heteroaryl or heterocyclyl ring; each R⁸ is independentlyselected from C₁₋₄ aliphatic; R⁹ is hydrogen; each R¹⁰ is independentlyselected from oxo, R¹¹, T-R¹¹, or V-T-R¹¹; each R¹¹ is independentlyselected from C₁₋₆ aliphatic, halo, —S(O)₂N(R⁷)₂, —OR⁷, —CN, —SR⁸,—S(O)₂R⁸, —C(O)R⁷, —CO₂R⁷, —N(R⁷)₂, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,—N(R⁷)CO₂R⁷, or —N(R⁷)C(O)N(R⁷)₂; T is a straight or branched C₁₋₄alkylene chain; V is —O—, —N(R⁷)—, —S—, —S(O)—, —S(O)₂—, —C(O)—, or—CO₂—; and R¹² is hydrogen, C₁₋₆ aliphatic, substituted or unsubstitutedphenyl, or substituted or unsubstituted benzyl.
 13. The compound ofclaim 12 where: R¹ is hydrogen, methyl, fluoro or amino; R² is chloro;R³ is hydrogen or alkoxy; n is zero or 2; R^(6b) is methyl; W is -Q-,-Q-C(O)— or —C(R⁹)₂—C(R⁹)(R¹²)—; Q is —C(R⁹)₂— or —C(R⁹)₂C(R⁹)₂—; G is—NR⁴R⁵, —N(R⁹)C(O)NR⁴R⁵, —N(R⁹)C(O)CF₃, —N(R⁹)CO(C₁₋₆ aliphatic), and—N(R⁹)CO(heterocyclyl) —N(R⁹)CO(heteroaryl), a 5-6 membered heterocyclylring, or a 5-6 membered heteroaryl, wherein each of the heteroaryl andheterocyclyl moieties of G is optionally substituted by 1-3 R¹⁰; R⁴ ishydrogen or C₁₋₆ aliphatic; R⁵ is hydrogen or C₁₋₆ aliphatic; each R⁷ isindependently selected from hydrogen or C₁₋₄ aliphatic, or two R⁷ on thesame nitrogen atom are taken together with the nitrogen to form a 5-6membered heteroaryl or heterocyclyl ring; each R⁸ is independentlyselected from C₁₋₄ aliphatic; R⁹ is hydrogen; each R¹⁰ is independentlyselected from oxo, R¹¹, T-R¹¹, or V-T-R¹¹; each R¹¹ is independentlyselected from C₁₋₆ aliphatic, halo, —S(O)₂N(R⁷)₂, —OR⁷, —CN, —SR⁸,—S(O)₂R⁸, —C(O)R⁷, —CO₂R⁷, —N(R⁷)₂, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,—N(R⁷)CO₂R⁷, or —N(R⁷)C(O)N(R⁷)₂; T is a straight or branched C₁₋₄alkylene chain; V is —O—, —N(R⁷)—, —S—, —S(O)—, —S(O)₂—, —C(O)—, or—CO₂—; and R¹² is hydrogen, C₁₋₆ aliphatic, phenyl, or benzyl.
 14. Acompound of formula (S)-III-A′:

or a pharmaceutically acceptable salt thereof, wherein: R¹ is hydrogen,methyl, fluoro or amino; R² is chloro; R³ is hydrogen or alkoxy; W is-Q-, -Q-C(O)— or —C(R⁸)₂—C(R⁸)(R¹²); Q is —C(R⁹)₂— or —C(R⁸)₂C(R⁹)₂—; Gis —NR⁴R⁵, —N(R⁹)C(O)NR⁴R⁵, —N(R⁹)C(O)CF₃, —N(R⁹)CO(C₁₋₆ aliphatic), and—N(R⁸)CO(heterocyclyl) —N(R⁹)CO(heteroaryl), a 5-6 membered heterocyclylring, or a 5-6 membered heteroaryl, wherein each of the heteroaryl andheterocyclyl moieties of G is optionally substituted by 1-3 R¹⁰; R⁴ ishydrogen or C₁₋₆ aliphatic; R⁵ is hydrogen or C₁₋₆ aliphatic; each R⁷ isindependently selected from hydrogen or C₁₋₄ aliphatic, or two R⁷ on thesame nitrogen atom are taken together with the nitrogen to form a 5-6membered heteroaryl or heterocyclyl ring; each R⁸ is independentlyselected from C₁₋₄ aliphatic; R⁹ is hydrogen; each R¹⁸ is independentlyselected from oxo, R¹¹, T-R¹¹, or V-T-R¹¹; each R¹¹ is independentlyselected from C₁₋₆ aliphatic, halo, —S(O)₂N(R⁷)₂, —CN, —SR^(a),—S(O)₂R⁸, —C(O)R⁷, —CO₂R⁷, —N(R⁷)₂, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,—N(R⁷)CO₂R⁷, or —N(R⁷)C(O)N(R⁷)₂; T is a straight or branched C₁₋₄alkylene chain; V is —O—, —N(R⁷)—, —S—, —S(O)—, —S(O)₂—, —C(O)—, or—CO₂—; and R¹² is hydrogen, C₁₋₆, aliphatic, phenyl, or benzyl.
 15. Thecompound of claim 14 having the formula (S)-III-A-a:

where R¹ is hydrogen, halo, methyl or amino; R² is hydrogen, methyl orhalo; R³ is hydrogen, halo, alkoxy, or (C₁₋₆ aliphatic)₂ amino; Ring Ais substituted by 0-2 R^(6b); R^(6b) is C₁₋₃ aliphatic; Q is —C(R⁹)₂— or—C(R⁹)₂C(R⁹)₂—; G is —NR⁴R⁵ or a substituted or unsubstituted 5-6membered heterocyclyl ring; R⁴ is hydrogen or C₁₋₆ aliphatic; R⁵ ishydrogen or a C₁₋₆ aliphatic group that is optionally substituted byhalo, —OR⁷, —CN, —SR⁸, —S(O)₂R⁸, —S(O)₂N(R⁷)₂, —C(O)R⁷, —CO₂R⁷, —N(R⁷)₂,—C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)CO₂R⁸, or —N(R⁷)C(O)N(R⁷)₂; each R⁷ isindependently selected from hydrogen or C₁₋₄ aliphatic, or two R⁷ on thesame nitrogen atom are taken together with the nitrogen to form a 5-6membered heteroaryl or heterocyclyl ring; each R⁸ is independentlyselected from C₁₋₄ aliphatic; and each R⁹ is independently hydrogen orC₁₋₃ aliphatic.
 16. A compound selected from the group consisting of:


17. A pharmaceutical composition comprising a compound of claim 1 and apharmaceutically acceptable carrier.
 18. A pharmaceutical compositioncomprising a compound of claim 16 and a pharmaceutically acceptablecarrier.
 19. A method of treating an IKK-mediated disease comprisingadministering to a patient in need of such treatment a therapeuticallyeffective amount of a compound of claim
 1. 20. The method of claim 19wherein the disease is an inflammatory disease or an immune-relateddisease.
 21. The method of claim 19 wherein the disease is selected fromthe group consisting of rheumatoid arthritis, asthma, psoriasis,psoriatic arthritis, chronic obstructive pulmonary disease, inflammatorybowel disease or multiple sclerosis.
 22. The method of claim 19 whereinthe disease is cancer.
 23. The method of claim 22 wherein the cancer isselected from lymphoma, multiple myeloma, osteolytic bone mestasis, heador neck cancer, lung cancer, prostate cancer or pancreatic cancer. 24.The method of claim 23 wherein the cancer is a lymphoma.
 25. A method ofinhibiting IRK in a patient in need thereof comprising administering tothe patient a compound of claim
 1. 26. A compound of formula 3a:

where R¹³ is halo, OH, OR¹⁵, or a carboxylic acid protecting group; R¹⁵is an aliphatic, aryl, heteroaryl, aralkyl, or heteroaralkyl; R¹⁴ is anamino protecting group, hydrogen or —W-G; W is -Q-, -Q-C(O)—,—C(R⁹)₂—C(R⁹)(R¹²)—, or —C(R⁹)₂—[C(R⁹)(R¹²)]₂—; Q is —C(R⁹)₂— or—C(R⁹)₂C(R⁹)₂—; G is —OH, —NR⁴R⁵, —N(R⁹)CONR⁴R⁵, —N(R⁹)SO₂ (C₁₋₃aliphatic), —N(R⁹)COCF₃, —N(R⁹)CO(C₁₋₆ aliphatic),—N(R⁹)CO(heterocyclyl), —N(R⁹)CO(heteroaryl) —N(R⁹)CO(aryl), a 3-7membered heterocyclyl ring, or a 5-6 membered heteroaryl, wherein eachof the heteroaryl, aryl and heterocyclyl moieties of G is optionallysubstituted by 1-3 R¹⁰; R⁴ is hydrogen, 3-7 membered heterocyclyl, orC₁₋₆ aliphatic; R⁵ is hydrogen, C_(1-E), aliphatic group or a 3-7membered heterocyclic ring having 1-2 ring heteroatoms selected from N,O, or S, wherein R⁵ is optionally substituted by halo, —CN, —SR⁸,—S(O)₂O, —S(O)₂N(R⁷)₂, —C(O)R⁷, —CO₂R⁷, —N(R⁷)₂, —C(O)N(R⁷)₂,—N(R⁷)C(O)R⁷, —N(R⁷)CO₂R⁸, or —N(R⁷)C(O)N(R⁷)₂; each R⁷ is independentlyselected from hydrogen or C₁₋₄ aliphatic, or two R⁷ on the same nitrogenatom are taken together with the nitrogen to form a 5-6 memberedheteroaryl or heterocyclyl ring; each R⁸ is independently selected fromC₁₋₄ aliphatic; each R⁹ is independently selected from hydrogen or C₁₋₃aliphatic; each R¹⁰ is independently selected from oxo, —R¹¹, -T-R¹¹, or-V-T-R¹¹; each R¹¹ is independently selected from C₁₋₆ aliphatic, halo,—S(O)₂N(R⁷)₂, —OR⁷, —CN, —SR⁸, —S(O)₂R⁸, —C(O)R⁷, —CO₂R⁷, —N(R⁷)₂,—C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)CO₂R⁷, or —N(R⁷)C(O)N(R⁷)₂; T is astraight or branched C₁₋₄ alkylene chain; V is —O—, —N(R⁷)—, —S—,—S(O)—, —S(O)₂—, —C(O)—, or —CO₂—; and R¹² is hydrogen or an amino acidside chain.
 27. The compound of claim 26 that is (S)-3a.
 28. A compoundof formula IV:

where R¹⁴ is an amino protecting group or hydrogen; R¹ is hydrogen,halo, C₁₋₃ aliphatic, amino, cyano, (C₁₋₃ alkyl)₁₋₂ amino, C₁₋₃ alkoxy,—CONH₂, —NHCOCF₃, or —CH₂NH₂; R² is hydrogen, halo, C₁₋₃ aliphatic,—CF₃; and R³ is hydrogen, halo, C₁₋₆ aliphatic, C₁₋₆ haloalkyl, C₁₋₆alkoxy, hydroxy, amino, cyano, or (C₁₋₆ alkyl)₁₋₂ amino.
 29. Thecompound of claim 28 that is (S)-IV.